Deimmunized lysostaphin and methods of use

ABSTRACT

Compositions comprising deimmunized lysostaphin and methods of using the same, e.g., to treat microbial infection in or on a subject, are provided.

This application claims the benefit of priority from U.S. PatentApplication Ser. No. 61/993,056, filed May 14, 2014, U.S. PatentApplication Ser. No. 62/003,256, filed May 27, 2014, U.S. PatentApplication Ser. No. 62/115,326, filed Feb. 12, 2015 and U.S. PatentApplication Ser. No. 62/155,079, filed Apr. 30, 2015, the contents ofwhich are incorporated herein by reference in their entireties.

This invention was made with government support under Grant Nos.1R21AI098122 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

INTRODUCTION Background

Staphylococcus aureus colonizes the skin and mucosal membranes of humansand animals, and together with the other members of the genusStaphylococcus, has been implicated in a diverse array of infections. S.aureus contains many virulence factors including surface proteinsdesignated as “microbial surface components recognizing adhesive matrixmolecules,” which facilitate attachment to surfaces and initiateinfection (Gordon & Lowy (2008) Clin. Infect. Dis. 46:S350-S359). S.aureus can also form biofilms (Donlan & Costerton (2002) Clin.Microbiol. Rev. 15:167-193), which allow it to evade both the immunesystem and antibiotics. Most strains have a polysaccharide capsule andsecrete a variety of enzymes that are used during infection to enhancebacterial spreading (Foster (2005) Nat. Rev. Microbiol. 3:948-958). S.aureus can also cause toxic shock syndrome, and studies have shown thatthe peptidoglycan and lipoteichoic acid of the S. aureus cell wall acttogether to cause toxic shock in rats (Kimpe, et al. (1995) Proc. Natl.Acad. Sci. USA 92:10359-10363).

Antibiotic resistance in staphylococci appeared after penicillin wasfirst used for treatment of staphylococcal infections. This developmentof resistance, which was present in over 80% of clinical isolates by thelate 1960s (Lowy (2003) J. Clin. Invest. 111:1265-1273), prompted thedevelopment of new, more potent drugs to combat the opportunisticpathogen. These efforts led to production of methicillin, a narrowspectrum penicillinase-resistant drug designed to alleviate the burdenof staphylococcal infections. However, it took only a year for the firstmethicillin-resistant S. aureus (MRSA) clinical isolates to bediscovered.

Initially, MRSA infections were only associated with prolonged hospitaltreatment and invasive surgical procedures, and were classified asHealth Care-Acquired MRSA (HCA-MRSA). However, in recent years, MRSA hasalso emerged as a community-acquired infection (CA-MRSA), which affectsgroups with high-intensity physical contact, such as competitiveathletes, military recruits, and children in daycare centers (Romano, etal. (2006) J. Athl. Train. 41:141-145; Kazakova, et al. (2005) New Engl.J. Med. 352:468-475; Zinderman, et al. (2004) Emerg. Infect. Dis.10:941-944; Adcock, et al. (1998) J. Infect. Dis. 178:577-580).

The S. aureus cell wall is composed of alternating polysaccharidesubunits of N-acetylglucosamine and N-acetylmuramic acid, wherein eachN-acetylmuramic acid is connected to a peptide chain. Cross-linking ofthe peptidoglycan is achieved by four major penicillin-binding proteins(PBP1, 2, 3 and 4) that connect the muropeptide chains via pentaglycineinterpeptide bridge. Methicillin resistance arose when S. aureusacquired the mecA gene, which encodes for penicillin-binding proteinPBP2A that has transpeptidase activity but lower affinity for penicillinand β-lactam antibiotics. Resistant cells still produce PBPs, but giventhe expression of PBP2A, peptidoglycan synthesis continues in thepresence of methicillin and other β-lactams (Hiramatsu, et al. (2001)Trends Microbiol. 9:486-493).

Lysostaphin is a glycyl-glycine zinc-dependent endopeptidase produced byStaphylococcus simulans, which selectively targets pentaglycineinterpeptide cross-bridges. The gene for lysostaphin has been isolatedand characterized. Genetic truncations have been made to remove the36-residue signal peptide and 224-residue long propeptide therebyfacilitating fusion to either an initiating methionine for intracellularexpression or an exogenous signal sequence, e.g., to permit thesecretion of a single species of lysostaphin into the periplasmic spaceof E. coli (See, e.g., US 2005/0118159). The mature, 247-residue enzymeis composed of N-terminal catalytic domain (138 amino acids), which isconnected to the C-terminal cell wall binding domain (92 amino acids)via an 18-residue linker (Lu, et al. (2013) Antimicrob. AgentsChemother. 57:1872-1881).

Lysostaphin has shown promise as a therapeutic agent for treatment of S.aureus infections. The protein has been shown to lyse staphylococcalstrains (Schindler & Schuhardt (1964) Proc. Natl. Acad. Sci. USA51:414421) and clinical isolates (Cropp & Harrison (1964) Can. J.Microbiol. 10:823-828), and demonstrated remarkable efficacy in animalmodels (Schuhardt & Schindler (1964) J. Bacteriol. 88:815-816;Schaffner, et al. (1967) Yale J. Biol. Med. 39:230-244; Goldberg, et al.(1967) Antimicrob. Agents Chemother. 7:45-53; Kokai-Kun, et al. (2007)J. Antimicrob. Ther. 60:1051-1059; Placencia, et al. (2009) Ped. Res.65:420-424; Climo, et al. (1998) Antimicrob. Agents Chemother.42:1355-60), including those of staphylococcal biofilms (Kokai-Kun, etal. (2009) J. Antimicrob. Ther. 64:94-100). In several of these studies,antibodies against lysostaphin were observed in animals subjected to thedrug for a prolonged period of time (Climo, et al. (1998) Antimicrob.Agents Chemother. 42:1355-1360). Similarly, human clinical trials withintranasal lysostaphin indicated a slight elevation in anti-lysostaphinantibody titer (Kokai-Kun (2012) in Antimicrobial Drug Discovery:Emerging Strategies (Tegos & Mylonakis, eds) Ch. 10, 147-165).

In an attempt to improve pharmacokinetics and reduce immunogenicity,lysostaphin has been linked to branched polyethylene glycol (PEG). WhilePEGylation reduced immunoreactivity, PEGylation of the enzymesignificantly reduced its activity (Walsh, et al. (2003) Antimicrob.Agents Chemother. 47:554-558). In addition, US 2008/0095756 describesthe deimmunization of the cell wall binding domain of lysostaphin.However, variants with a deimmunized catalytic domain are not described.

SUMMARY OF THE INVENTION

This invention is a deimmunized lysostaphin having a mutation at one ormore of Ser124, Ser122, Asn121, Arg118, Ile99, Lys95, Tyr93, Leu83,Lys46, Ile41, Asn13, Asn12 of SEQ ID NO:49. In one embodiment, thelysostaphin is aglycosylated. In another embodiment, the mutation isSer124Gly, Ser122Asp, Asn121Gly, Arg118Thr, Ile99Gln, Lys95Glu,Tyr93His, Leu83Met, Lys46His, Ile41Glu, Asn13His, Asn12Gly, or acombination thereof. In a further embodiment, the deimmunizedlysostaphin further includes one or more amino acid substitutions in theC-terminal binding domain. A pharmaceutical composition containing thedeimmunized lysostaphin and an antibiotic is provided, as is a methodpreventing or treating a microbial infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E show a sequence alignment of lysostaphin variants of thepresent invention.

FIG. 2 shows an epitope map of the lysostaphin catalytic domain. Thetotal number of predicted binding events is plotted against thelysostaphin primary sequence. Using EpiMatrix, epitopes were predictedfor MHC II alleles DRB*0101, 0301, 0401, 0701, 0801, 1101, 1301, and1501. The maximum score is 8 and represents an epitope that is predictedto bind all 8 alleles. Such an epitope was observed at position 116. Thesites of EpiSweep mutations are indicated with arrows and residuenumbers. Epitope groups are divided into five distinct clusters.Mutations found to be detrimental for lysostaphin expression andactivity that were later dropped (Phe38Gly and Ser124Tyr) are alsoindicated.

FIG. 3 shows an aggregate of immunogenicity scores calculated forfull-length designs. Each peptide in a design was evaluated as strong(IC₅₀<1 μM), moderate (1 μM<IC₅₀<10 μM), or weak (10 μM<IC₅₀<100 μM)binder to the eight MHC class II alleles. Strong, moderate and weakbinders were then summed to obtain the aggregate score shown in thefigure. The line with an “a” indicates the number of strong binders inthe wild type lysostaphin catalytic domain, while the line with a “b”shows the number of moderate binders. Numbers on the bars represent themutational load of each design. *Indicates reverted designs.

FIGS. 4A-4C shows in vivo efficacy and immunogenicity analysis of Flex 5and Flex 9 variants. FIG. 4A, Bacterial burden in the lungs of C57Bl/6mice following infection with S. aureus and treatment with wild-typeLST, variant Flex 5, variant Flex 9, or a PBS control. N=6 per group.FIG. 4B, HUMI mice (all humanized from a single donor) were immunizedsubcutaneously with either wild-type LST (WT), variant Flex 5, orvariant Flex 9, and splenocytes were harvested and restimulated ex vivowith the same protein or DMSO. Proliferation was measured as uptake oftritiated thymidine. N=4 per group, pooled and measured in triplicate.FIG. 4C, Transgenic DR4 mice were immunized with multiple subcutaneousinjections of wild-type LST. Following the final boost, mice wereallowed to recover for 20 weeks, divided into two groups, andrechallenged with either wild-type LST or variant Flex 5. Splenocyteswere harvested and restimulated ex vivo with the rechallenge protein orDMSO, and proliferation was measured as uptake of tritiated thymidine.N=5 per group, pooled and measured in triplicate. Statisticalsignificance was assessed by one way ANOVA (FIG. 4A) or two way ANOVA(FIGS. 4B and 4C). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

FIG. 5 shows the predicted DR4 T cell epitopes for variants Lib5 (top),Opt4 (middle), and LST^(WT) (bottom). Nonamer epitopes are shown ashorizontal lines at the corresponding position in the LST amino acidsequence (X-axis). LST^(WT) contains 16 putative epitopes, Opt4 contains5, and Lib5 contains only three. Regions of overlapping epitope densityin LST^(WT) are highlighted with filled blocks on the x-axis. Positionsof deimmunizing mutations in Lib5 (top) and in Opt4 (middle) are shownas filled vertical bars. The mutation (M) and epitope (E) counts foreach design are indicated at right.

FIGS. 6A and 6B show the global epitope map of the lysostaphin catalyticand cell wall binding domain, respectively. Epitopes were predicted forHLA alleles DRB1*0101, 0301, 0401, 0701, 0801, 1101, 1301, and 1501.Nine residue peptide epitopes are indicated by solid lines (1-2%threshold) and dashed lines (3-5% threshold). The Lysostaphin catalyticdomain primary sequence is indicated on the x-axis, and the HLA allelesassociated with each peptide epitope are shown on the y-axis. Higherrisk regions for designability analysis are marked at top with filledboxes. The box with the broken line represents a moderate risk regionthat was also redesigned during development of the deimmunized Flex 9deimmunized catalytic domain.

FIG. 7 shows the minimum inhibitory concentration (MIC) for F11, F12 andF13 against MRSA strain USA400.

FIG. 8 shows the in vivo efficacy of the F11 variant in C57BL/6 mice.Mice were challenged with an intraperitoneal injection of 2×10⁸ MRSAstrain USA400, and 1 hour later the mice were treated with 100 μgwild-type LST, F11 or PBS. The percent survival is shown.

DETAILED DESCRIPTION OF THE INVENTION

It has now been shown that the catalytic domain of lysostaphin can bedeimmunized without significantly altering enzymatic activity. Inparticular, EpiSweep analysis of lysostaphin was used to identify MHC IIbinding events. Deimmunized lysostaphin variants containing mutations inthe catalytic domain were generated, expressed, purified and shown tohave an activity level comparable to commercially-sourced lysostaphin.By combining mutations in the catalytic domain with mutations in thecell wall binding domain, this invention provides a fully deimmunizedlysostaphin variant and methods of using the same to treat a microbialinfection.

As used herein, the term “deimmunized” when used in reference tolysostaphin, relates to lysostaphin (e.g., lysostaphin variants,derivatives and/or homologues thereof), wherein the specific removaland/or modification of highly immunogenic regions or residues hasoccurred. The term “deimmunized” is well-known in the art and, amongother things, has been employed for the removal of T-cell epitopes fromother therapeutic molecules including antibodies (See, e.g., WO 98/52976or WO 00/34317).

Humoral antibody formation requires the cooperation of helper T-cellswith antigen-specific B-cells. To reduce immunogenicity of a molecule,one approach is to reduce the ability of the antigen to interact withand stimulate B-cells and/or reduce their ability to stimulate helperT-cells. The identification of B-cell epitopes is problematic, however,given the fact that they are of indeterminate length, and oftendependent on the tertiary structure of the target antigen. In contrast,T cell epitopes are short (9-15 amino acid), linear peptides (See, e.g.,Doytchinova & Flower (2006) Mol. Immunol. 43(13):2037-44). In addition,evidence suggests that reduction of T-cell activation is easier toachieve and has the ability to greatly impact antibody production (see,e.g., Tangri, et al. (2005) J. Immunol. 174:3187-3196). The amino acidsequences that include the antigenic determinants that stimulate T-cellsare referred to as T-cell epitopes and are displayed in the context ofmajor histocompatibility complex (MHC) molecules on antigen presentingcells. Altering the ability of T cell epitopes to bind MHC molecules(e.g., by inhibiting the binding of the epitope to the MHC molecule,altering the affinity between the epitope and the MHC molecule, alteringthe epitope in a manner such that the epitope's orientation is alteredwhile within the binding region of the MHC molecule, or altering theepitope in such a way that its presentation by the MHC molecule isaltered) has the potential to render the altered epitopes unable to orless able to stimulate an immunogenic response (e.g., stimulate helperT-cells and B cell responses). Accordingly, using the methods describedherein, epitopes of lysostaphin were identified and subsequently alteredin an effort to reduce the immunogenicity of lysostaphin and its abilityto induce humoral antibody responses.

Thus, deimmunization involves the identification, modification and/orremoval of T-cell epitopes, preferably helper T-cell epitopes. In thiscontext, the term T-cell epitope relates to T-cell epitopes (i.e., smallpeptides) that are recognized by T-cells in the context of MHC class Iand/or class II molecules. Methods for the identification of T-cellepitopes are known in the art (see, e.g., WO 98/52976, WO 00/34317, andUS 2004/0180386). Various methods of identification include, but are notlimited to, peptide threading, peptide-MHC binding, human T-cell assays,analysis of cytokine expression patterns, ELISPOT assays, class IItetramer epitope mapping, search of MHC-binding motif databases and theadditional removal/modification of T-cell epitopes. In particularembodiments, a structure-guided deimmunization approach, such as thatemployed by the EpiSweep method, is used. EpiSweep integratesstructure-based protein design, sequence-based protein deimmunization,and algorithms for finding the Pareto frontier of a design space(Parker, et al. (2013) J. Comput. Biol. 20:152-65).

Having identified T cell epitopes by application of the above-recitedtechnologies, the epitopes can be eliminated, substituted and/ormodified from lysostaphin or fragment(s) thereof (e.g., the catalyticdomain) by one or more amino acid substitutions within an identified MHCbinding peptide as further described herein. In some embodiments, one ormore amino acid substitutions are generated that eliminate or greatlyreduce binding to MHC class I and/or class II molecules, oralternatively, altering the MHC binding peptide to a sequence thatretains its ability to bind MHC class I or class II molecules but failsto trigger T cell activation and/or proliferation.

Mature lysostaphin has been shown to have two functional domains, aC-terminal domain of 92 residues that binds the S. aureus outer cellwall and the N-terminal active site having endopeptidase activity (Baba& Schneewind (1996) EMBO J. 15:4789-4797). Lysostaphin has not beensuccessfully crystallized in part due to the differing solventcharacteristics of its two separate domains. However, using the insilico methods described herein, highly functional lysostaphin proteins,including various combinations of mutations, have been produced. Mutableamino acids at each position in the catalytic domain (except active siteresidues) were selected lysostaphin variants were produced that werepredicted to have lower immunogenicity while retaining stability. Eachmutation was evaluated for expression and activity. Only the mutationsthat were deemed satisfactory in both regards were selected, and thedeimmunization process was repeated again. After the appropriate energyminimizations of the resulting plans, designs with the best predictedenergy scores were chosen and experimentally tested. Lysostaphinvariants that were capable of being expressed were then purified andfurther characterized for activity, stability and immunogenicity.

Accordingly, the present invention provides a variety of lysostaphinvariants, including modification (e.g., mutations such as amino acidsubstitutions) of immunogenic epitopes, which retain activity whileconcurrently displaying reduced immunogenicity. As used herein, the term“lysostaphin” refers to amino acid sequence and/or nucleic acid sequenceencoding full length lysostaphin or portion thereof, any lysostaphinmutant or variant (e.g., lysostaphin of any one of SEQ ID NOs: 1-48 or218-220), any lysostaphin truncation (e.g., in which one or more aminoacids have been removed from the protein's amino terminus, carboxyterminus, or both), and any recombinantly expressed lysostaphin protein,that retains the proteolytic ability, in vitro and in vivo, ofproteolytic attack against glycine-containing bridges in the cell wallpeptidoglycan of staphylococci. Lysostaphin variants (e.g., deimmunizedlysostaphin described herein) may also be expressed in a truncated form.Modified full-length lysostaphin or lysostaphin variants may begenerated by post-translational processing of the protein (either byenzymes present in a host cell strain or by means of enzymes or reagentsintroduced at any stage of the process) or by mutation of the structuralgene. Lysostaphin variants, as describe herein, may include deletion,insertion, domain removal, point and replacement/substitution mutations.

The present invention is not limited to any particular lysostaphinvariant. Indeed, a variety of variants are provided by the presentinvention including, but not limited to, those described in the Examplesand depicted in FIGS. 1A-1E. In some embodiments, a lysostaphin varianthas a single amino acid substitution (e.g., any one of the amino acidsubstitutions described herein) when compared with the wild-typesequence: AATHEHSAQWLNNYKKGYGYGPYPLGINGGMHYGVDFFMNIGTPVKAISSGKIVEAGWSNYGGGNQIGLIENDGVHRQWYMHLSKYNVKVGDYVKAGQIIGWSGSTGYSTAPHLHFQRMVNSFSNPTAQDPMPFLKSAGYGKAGGTVTPTPNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQSGVLKAGQTIHYDEVMKQDGHVWVGYTGNSGQRIYLPVRTWNKSTN TLGVLWGTIK(SEQ ID NO:49). In some embodiments, a lysostaphin variant has two aminoacid substitutions when compared with the wild-type sequence. In otherembodiments, a lysostaphin variant has three amino acid substitutionswhen compared with the wild-type sequence. In further embodiments, alysostaphin variant has four or more amino acid substitutions whencompared with the wild-type sequence. In certain embodiments, alysostaphin variant has one or more amino acid substitutions in thecatalytic domain. In some embodiments, a lysostaphin mutant has amutation at Ser124, Ser122, Asn121, Arg118, Ile99, Lys95, Tyr93, Leu83,Lys46, Ile41, Asn13, Asn12 or a combination thereof. In someembodiments, a lysostaphin variant has one or a combination of thefollowing mutations: Ser124Gly, Ser122Asp, Asn121Gly, Arg118Thr,Ile99Gln, Lys95Glu, Tyr93His, Leu83Met, Lys46His, Ile41Glu, Asn13His,and Asn12Gly. In other embodiments, a lysostaphin variant also has oneor more amino acid substitutions in the C-terminal binding domain. Insome embodiments, a lysostaphin variant has a C-terminal binding domainmutation at Asn236, Arg186, Ala169, Ser166, Tyr160 or a combinationthereof. In some embodiments, a lysostaphin variant has one or acombination of the following mutations in the C-terminal binding domainmutation: Asn236Asp, Arg186Thr, Ala169Gly, Ser166Asn and Tyr160His.Other suitable amino acid substitutions in the C-terminal binding domaininclude, but are not limited to those disclosed in US 2008/0095756.

Similarly, the present invention is not limited to any particular typeof mutation. Mutations of this invention include, but not limited to,amino acid exchange(s), insertion(s), deletion(s), addition(s),substitution(s), inversion(s) and/or duplication(s). Thesemutations/modification(s) also include conservative and/or homologueamino acid exchange(s). Guidance concerning how to makephenotypically/functionally silent amino acid substitution has beendescribed (see, e.g., Bowie (1990), Science 247:1306-1310).

The present invention also provides lysostaphin variants having an aminoacid sequence that is at least 60%, more preferably at least 70%, morepreferably at least 80%, more preferably 90%, more preferably at least95% and most preferably 99% identical or homologous to the polypeptidesequences shown in FIG. 1 (SEQ ID NOs: 1-48) or in SEQ ID NO:218-220.

In some embodiments, a lysostaphin variant of the present inventionelicits less than 90%, more preferably less than 80%, more preferablyless than 70%, more preferably less than 60%, more preferably less than50%, more preferably less than 40%, more preferably less than 30%, morepreferably less than 20%, and even more preferably less than 10% of theimmune response (e.g., as measured by anti-lysostaphin antibody titers)elicited by non-deimmunized lysostaphin.

In some embodiments, the present invention provides a plasmid harboringa nucleic acid sequence encoding deimmunized a lysostaphin variant. Incertain embodiments, the plasmid is an expression vector harboring anucleic acid sequence encoding a lysostaphin variant (e.g., thatdisplays bactericidal activity and reduced immunogenicity and). In someembodiments, the lysostaphin variant is expressed as a fusion protein,e.g., fused to sequences that facilitate purification (e.g., histidinestretches). In some embodiments, an expression vector of the presentinvention harbors a nucleic acid sequence encoding a deimmunizedlysostaphin variant having an amino acid sequence as set forth in SEQ IDNO:1-48 (FIGS. 1A-1E) or SEQ ID NO:218-220.

In addition to lysostaphin variant nucleic acids, a plasmid of thisinvention may also include regulatory sequences, e.g., promoters,transcriptional enhancers and/or sequences that allow for inducedexpression of lysostaphin variants. For example, one suitable induciblesystem is a tetracycline-regulated gene expression system (see, e.g.,Gossen & Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossenet al. (1994) Trends Biotech. 12:58-62). In some embodiments, theinducible system is an isopropyl-b-D-thiogalactoside (IPTG)-induciblepromoter.

Using expression plasmids, the lysostaphin variant of this invention canbe produced by a number of known methods. For example, the lysostaphinvariant can be expressed and isolated from Bacillus sphaericus (U.S.Pat. No. 4,931,390); Lactococcus lactis NICE expression system(NIsin-Controlled gene Expression) (Mierau, et al. (2005) Microb. CellFact. 4:1-9); pET23b(+) and pBAD/Thio-TOPO E. coli expression systems(Szweda, et al. (2005) J. Biotechnol. 117:203-213); BL21 (DE-3) E. coli(Sharma, et al. (2006) Prot. Exp. Purific. 45:206-215); or Pichiapastoris, as described herein and elsewhere for the production oftherapeutic proteins (Gasser, et al. (2013) Future Microbiol. 8:191-208;Walsh (2010) Nature Biotechnol. 28:917-924; Shekhar (2008) Chem. Biol.15:201-202; Meyer, et al. (2008) Bioproc. Internat. 6:10-21). Inparticular embodiments, the lysostaphin variant of this invention isobtained by expression in P. pastoris, which is characterized withefficient and selective secretion, high protein titers, and high celldensity cultivations (Vogl, et al. (2013) Curr. Opin. Biotechnol.24:1094-1101). Furthermore, P. pastoris is considered as a safe (GRAS)organism, has several signal sequences that can be used for proteinsecretion, and has one of the strongest promoters known (AOX). Becauseit allows for protein secretion directly into media, P. pastoris greatlysimplifies protein recovery and downstream purification.

The lysostaphin variants of this invention can be purified by a numberof known methods. For example, due to its high positive charge,lysostaphin has been purified from bacterial hosts such as S. simulans,B. sphaericus, or L. lactis using a cation exchange step (Recsei, et al.(1990) supra; Mierau, et al. (2005) supra; Fedorov, et al. (2003)Biochemistry (Moscow) 68:50-53). When expressed in E. coli, lysostaphinhas been purified using affinity chromatography (Szweda, et al. (2005)supra; Sharma, et al. (2006) supra).

Lysostaphin activity can be determined in several different ways:minimum inhibitory concentration (MIC), minimum bactericidalconcentration (MBC), disk diffusion, and turbidity reduction (Kusuma &Kokai-Kun (2005) Antimicrob. Agents Chemother. 49:3256-3263). The MICassay is performed to obtain the minimum concentration of lysostaphinthat is necessary to prevent growth of S. aureus cells, while MBC assayis usually done after a MIC assay to determine the minimal concentrationof the drug necessary to kill S. aureus. MIC assays are considered to bethe golden standard of determining the value of a therapeutic. The diskdiffusion assay is conducted to measure activity by determining thediameter of zone of clearance that is created when alysostaphin-containing disk is placed on a lawn of S. aureus and allowedto diffuse into the plate media over time. The turbidity reduction assayinvolves measuring the decrease in optical absorbance of S. aureusculture over time as lysis of the cells progresses (Schindler &Schuhardt (1964) Proc. Natl. Acad. Sci. USA 51:414-421).

Protein stability can be determined using several different methods.Three well-established methods for measuring thermostability include,e.g., differential scanning calorimetry (DSC), differential scanninglight scattering (DSLS), and differential scanning fluorimetry (DSF).All methods are based on determining the rate of protein unfolding withincreasing temperature, which is a measure of protein stability. Forinstance, if a small increase in temperature results in proteinunfolding, the protein is not considered to be very stable. DSC directlymeasures the heat absorption associated with thermal denaturation andhas been proven to be sufficiently quantitative for evaluation ofstability of protein therapeutics (Wen, et al. (2011) J. Pharmaceut.Sci. 101:955-964). The DSLS method measures protein stability based onthe assumption that proteins denature irreversibly as they are exposedto increasing temperatures. Using light-scattering, this method monitorsthe aggregation that occurs as a consequence of denaturation. In DSF, afluorescent dye is used that fluoresces upon binding hydrophobicresidues. As temperature increases, the protein starts to unfold andexposes the hydrophobic residues found in its core, causing an increasein the fluorescent signal. This increase in signal is monitored over arange of temperatures and is used to determine the Tm value.

To assess immunogenicity, in vitro and in vivo models have beengenerated. Since MHC molecules play an important role in T celldependent immune responses, in vitro assays can be used to test theability of a peptide to bind MHC (Salvat, et al. (2014) J. Vis. Exp.85). In addition, several animal models such as rats, mice, andnon-human primates are currently used in the pre-clinical evaluation ofprotein therapeutics, wherein the closer a model is to humans, the moreaccurate it will be at predicting unwanted antibody production inpatients (Brinks, et al. (2013) Pharma. Res. 30:1719-1728).

In some embodiments, the present invention provides a pharmaceuticalcomposition containing a lysostaphin variant of the present invention.For example, in some embodiments, the present invention provides acomposition containing a lysostaphin variant and a pharmaceuticallyacceptable carrier. In certain embodiments, the present inventionprovides a lysostaphin variant (e.g., deimmunized lysostaphin) of use ina pharmaceutical composition for treatment or prevention ofstaphylococcal infection (e.g., of the skin, of a wound, or of an organ)or as a therapy for various active S. aureus infections. In preferredembodiments, a pharmaceutical composition of the present inventionincludes a therapeutically effective amount of a lysostaphin of theinvention, together with a pharmaceutically acceptable carrier. Thepresent invention is not limited by the types of pharmaceuticallyacceptable carrier utilized. Indeed, a variety of carriers are wellknown in the art including, but not limited to, sterile liquids, such aswater, oils, including petroleum oil, animal oil, vegetable oil, peanutoil, soybean oil, mineral oil, sesame oil, and the like. Salinesolutions, aqueous dextrose, and glycerol solutions can also be employedas liquid carriers, particularly for solution preparations forinjection. Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, 18th Edition.

A therapeutically effective amount is an amount of lysostaphin variantreasonably believed to provide some measure of relief, assistance,prophylaxis, or preventative effect in the treatment of infection. Atherapeutically effective amount may be an amount believed to besufficient to block a bacterial colonization or infection. Similarly, atherapeutically effective amount may be an amount believed to besufficient to alleviate (e.g., eradicate) an existing bacterialinfection. A pharmaceutical composition of the present invention may beparticularly useful in preventing, ameliorating and/or treatingbacterial infection.

The compositions of the invention may be administered locally (e.g.,topically) or systemically (e.g., intravenously). Preparations forparenteral administration include sterile aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous solventsare propylene glycol, polyethylene glycol, vegetable oils such as oliveoil, and injectable organic esters such as ethyl oleate. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Parenteral vehiclesinclude sodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's, or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers (such as thosebased on Ringer's dextrose), and the like. Preservatives and otheradditives may also be present such as, for example, antimicrobials,anti-oxidants, chelating agents, and inert gases and the like.Furthermore, the pharmaceutical composition of the invention maycomprise further agents depending on the intended use of thepharmaceutical composition.

In accordance with this invention, the terms “treatment”, “treating” andthe like are used herein to generally mean obtaining a desiredpharmacological and/or physiological effect. The effect may beprophylactic in terms of completely or partially preventing an infectionand/or may be therapeutic in terms of completely or partially treating(e.g., eradicating) a bacterial infection. The term “treatment” as usedherein includes preventing bacterial infection from occurring in asubject (e.g., that may be predisposed to infection (e.g., nosocomialinfection) but has not yet been diagnosed as having infection);inhibiting bacterial infection; and/or (c) relieving infection (e.g.,completely or partially reducing the presence of bacteria responsiblefor infection.

Staphylococcal infections, such as those caused by S. aureus, are asignificant cause of morbidity and mortality, particularly in settingssuch as hospitals, schools, and infirmaries. Patients particularly atrisk include infants, the elderly, the immunocompromised, theimmunosuppressed, and those with chronic conditions requiring frequenthospital stays. Patients also at risk of acquiring staphylococcalinfections include those undergoing inpatient or outpatient surgery,those within an Intensive Case Unit (ICU), on continuous hemodialysis,with HIV infection, with AIDS, burn victims, people with diminishedimmunity (e.g., resulting from drug treatment or disease), thechronically ill or debilitated patients, geriatric subjects, infantswith immature immune systems, and people with intravascular (e.g.,implanted) devices. Thus, in some embodiments, a composition containinga lysostaphin variant is administered to any one of these types ofsubject as well as to other subjects that have or are susceptible tobacterial infection (e.g., caused by S. aureus or S. epidermidis).

In some embodiments, a lysostaphin variant of the present invention isformulated as either an aqueous solution, semi-solid formulation, or drypreparation (e.g., lyophilized, crystalline or amorphous, with orwithout additional solutes for osmotic balance) for reconstitution.Formulations may be in, or reconstituted in, for example, a non-toxic,stable, pharmaceutically acceptable, aqueous carrier medium, at a pH ofabout 3 to 8, typically 5 to 8, for administration by conventionalprotocols and regimes or in a semi-solid formulation such as a cream.Delivery can be via, for example, ophthalmic administration, intravenous(iv), intramuscular, subcutaneous or intraperitoneal routes orintrathecally or by inhalation or used to coat medical devices,catheters and implantable devices, or by direct installation into aninfected site so as to permit blood and tissue levels in excess of theminimum inhibitory concentration (MIC) of the active agent to beattained (e.g., to effect a reduction in microbial titers in order tocure, alleviate or prevent an infection). In some embodiments, theantimicrobial agent is formulated as a semi-solid formulation, such as acream (e.g., that is used in a topical or intranasal formulation).

Furthermore, the lysostaphin variant can be co-administered,simultaneously or alternating, with other antimicrobial agents so as tomore effectively treat an infectious disease. Formulations may be in, orbe reconstituted in, semi-solid formulations for topical, ophthalmic, orintranasal application, liquids suitable for ophthalmic administration,bolus iv or peripheral injection or by addition to a larger volume ivdrip solution, or may be in, or reconstituted in, a larger volume to beadministered by slow iv infusion. For example, a lysostaphin variant canbe administered in conjunction with antibiotics that interfere with orinhibit cell wall synthesis, such as penicillins, nafcillin, and otheralpha- or beta-lactam antibiotics, cephalosporins such as cephalothin,aminoglycosides, sulfonamides, antifolates, macrolides, quinolones,glycopepetides such as vancomycin and polypeptides. In some embodiments,a lysostaphin variant is administered in conjunction with one or moreantibiotics that inhibit protein synthesis (e.g., aminoglycosides suchas streptomycin, tetracyclines, and streptogramins). The presentinvention is not limited by the type of agent co-administered withdeimmunized lysostaphin. Indeed, a variety of agents may beco-administered including, but not limited to, those agents described inU.S. Pat. Nos. 6,028,051, 6,569,830, and 7,078,377, each of which ishereby incorporated by reference in its entirety. In some embodiments, alysostaphin variant is administered with monoclonal antibodies; othernon-conjugated antibacterial enzymes such as lysostaphin, lysozyme,mutanolysin, and cellozyl muramidase; peptides (e.g., defensins); andlantibiotics (e.g., nisin); or any other lanthione-containing molecules(e.g., subtilin).

Agents co-administered with a lysostaphin variant may be formulatedtogether with the lysostaphin variant as a fixed combination or may beused extemporaneously in whatever formulations are available andpractical and by whatever routes of administration are known to provideadequate levels of these agents at the sites of infection.

In preferred embodiments, lysostaphin variants according to the presentinvention possess at least a portion of the antimicrobial activity ofthe corresponding non-deimmunized antimicrobial agent. A lysostaphinvariant of the present invention may be administered in increaseddosages and/or at less frequent intervals due to the decreasedimmunogenicity. In some embodiments, a lysostaphin variant retains atleast 10% of the activity of the non-deimmunized antimicrobial agent. Insome embodiments, a lysostaphin variant retains at least 20% of theactivity of the non-deimmunized antimicrobial agent. In someembodiments, a lysostaphin variant retains at least 30% of the activityof the non-deimmunized antimicrobial agent. In some embodiments, alysostaphin variant retains at least 40% of the activity of thenon-deimmunized antimicrobial agent. In some embodiments, a lysostaphinvariant retains at least 50% of the activity of the non-deimmunizedantimicrobial agent. In some embodiments, a lysostaphin variant retainsat least 60% of the activity of the non-deimmunized antimicrobial agent.In some embodiments, a lysostaphin variant retains at least 70% of theactivity of the non-deimmunized antimicrobial agent. In someembodiments, a lysostaphin variant retains at least 80% of the activityof the non-deimmunized antimicrobial agent. In some embodiments, alysostaphin variant retains at least 90% of the activity of thenon-deimmunized antimicrobial agent. In some embodiments, a lysostaphinvariant retains 90% or more (e.g., 95%, 97%, 99% or more) of theactivity of the non-deimmunized antimicrobial agent.

Suitable dosages and regimes of a deimmunized lysostaphin may vary withthe severity of the infection and the sensitivity of the infectingorganism and, in the case of combination therapy, may depend on theparticular agent (e.g., anti-staphylococcal agent) co-administered.Dosages may range from about 0.05 to about 500 mg/kg/day (e.g., in someembodiments, range from 0.1-10 mg/kg/day, in some embodiments, rangefrom 10-100 mg/kg/day, in some embodiments, range from 100-200mg/kg/day, in some embodiments, range from 200-400 mg/kg/day, in someembodiments, range from 400-500 mg/kg/day), although higher (e.g.,500-1000 mg/kg/day) or lower (e.g., 0.1-0.5 mg/kg/day doses may beprovided, given as single or divided doses, or given by continuousinfusion. In some embodiments, deimmunized lysostaphin is administeredonce a day, twice a day, three times a day or more frequently (e.g.,four or more times a day). In some embodiments, deimmunized lysostaphinis administered once a week, twice a week, or every other day. In someembodiments, deimmunized lysostaphin is administered once every otherweek, once a month, once every two months, once every three months, onceevery four months, once every five months, once every six months, onceevery 9 months, once every year or less frequently.

In certain embodiments, a deimmunized lysostaphin of this invention isaglycosylated. Aglycosylation can be carried out as described here, andcan include mutations at residues Ser126 and/or Thr127. Exemplarymutations include, Ser126Pro and Thr127Ala.

In some embodiments, a deimmunized lysostaphin of the present inventionmay be further modified in order to further decrease immunogenicity ofthe lysostaphin molecule while retaining antimicrobial activity. Forexample, in some embodiments, a deimmunized lysostaphin is conjugated toa water soluble polymer. The present invention is not limited by thetype of water soluble polymer to which a deimmunized lysostaphin isconjugated. Indeed, a variety of water soluble polymers may be usedincluding, but not limited to, poly(alkylene oxides), polyoxyethylatedpolyols and poly(vinyl alcohols). Poly(alkylene oxides) include, but arenot limited to, polyethylene glycols (PEGs), poloxamers and poloxamines.The present invention is not limited by the type of conjugation used(e.g., to connect a deimmunized lysostaphin to one or more water-solublepolymers (e.g., PEG)). In some embodiments, a poly(alkylene oxide) isconjugated to a free amino group via an amide linkage (e.g., formed froman active ester such as the N-hydroxysuccinimide ester) of thepoly(alkylene oxide). In some embodiments, an ester linkage remains inthe conjugate after conjugation. In some embodiments, linkage occursthrough a lysine residue present in the deimmunized lysostaphinmolecule. In some embodiments, conjugation occurs through ashort-acting, degradable linkage. The present invention is not limitedby the type of degradable linkage utilized. Indeed, a variety oflinkages are contemplated to be useful in the present inventionincluding, but not limited to, physiologically cleavable linkagesincluding ester, carbonate ester, carbamate, sulfate, phosphate,acyloxyalkyl ether, acetal, and ketal linkages. In some embodiments,deimmunized lysostaphin is conjugated to PEG utilizing any of themethods, reagents and/or linkages described in U.S. Pat. Nos. 4,424,311;5,672,662; 6,515,100; 6,664,331; 6,737,505; 6,894,025; 6,864,350;6,864,327; 6,610,281; 6,541,543; 6,515,100; 6,448,369; 6,437,025;6,432,397; 6,362,276; 6,362,254; 6,348,558; 6,214,966; 5,990,237;5,932,462; 5,900,461; 5,739,208; 5,446,090 and 6,828,401; and WO02/02630 and WO 03/031581. In some embodiments, a deimmunizedlysostaphin-water soluble polymer conjugate of the present invention isproduced by a third party (e.g., NEKTAR, San Carlos, Calif.). In someembodiments, the conjugate includes a cleavable linkage present in thelinkage between the polymer and deimmunized lysostaphin (e.g., such thatwhen cleaved, no portion of the polymer or linkage remains on thedeimmunized lysostaphin molecule). In some embodiments, the conjugateincludes a cleavable linkage present in the polymer itself (e.g., suchthat when cleaved, a small portion of the polymer or linkage remains onthe deimmunized lysostaphin molecule).

In some embodiments, a deimmunized lysostaphin of the present inventionis used for the treatment and/or prevention of a biofilm (e.g., asdescribed in US 2003/0215433 and WO 03/082148). In other embodiments, adeimmunized lysostaphin of the present invention is used in theprevention and/or treatment of a microbial infection, includingbacterial infections by members of the genus Staphylococcus. Accordingto such methods, a subject in need of treatment (e.g., a subject with orat risk of developing an S. aureus infection) is administered aneffective amount of a deimmunized lysostaphin so that the microbialinfection is prevented or treated. Subjects benefiting from thistreatment include those exhibiting clinical signs or symptoms of aninfection, a subject exposed to a bacterium (e.g., S. aureus), or asubject suspected of being exposed to a bacterium (e.g., S. aureus).Effective treatment will result in a decrease, attenuation, inhibitionor amelioration of the well-known signs or symptoms of infection. Insome embodiments, treatment includes nasal applications, e.g., asdescribed in US 2003/0211995; or topical applications, e.g., asdescribed in US 2004/0192581.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular deimmunized lysostaphinemployed, the route of administration, the time of administration, therate of excretion or metabolism of the particular deimmunizedlysostaphin, the duration of the treatment, other drugs, compoundsand/or materials used in combination with the particular deimmunizedlysostaphin employed, the age, sex, weight, condition, general healthand prior medical history of the patient being treated, and like factorswell known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of a deimmunized lysostaphin at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved. Thisis considered to be within the skill of the artisan.

Effective doses can also be determined in an art-recognized model of S.aureus infection. There are many different in vivo model systems thatcan be used by one of skill in the art to further demonstrate efficacyand aid in identification of doses that will be both safe and effectivein humans. Such animal model systems are well-accepted and used duringdevelopment of new human pharmaceuticals. Examples of such model systemsinclude, but are not limited to, a guinea pig model of S. aureus woundinfection (Kernodle & Kaiser (1994) Antimicrob. Agents Chemother.38:1325-1330); a rabbit model of S. aureus abscess in rabbits(Fernandez, et al. (1999) Antimicrob. Agent Chemother. 43:667-671); amouse model of S. aureus skin infection (Gisby & Bryant (2000)Antimicrob. Agents Chemother. 44:255-260); a mouse model of deep dermalS. aureus infection (Godin, et al. (2005) J. Antimicrob. Chemother.55:989-994); and a mouse intraperitoneal infection model (Patel, et al.(2004) Antimicrob. Agents Chemother. 48:4754-4761). In such models,therapeutics can be tested against infections where the infectionestablished is from inoculation of the animal with various strains of S.aureus. Demonstration of efficacy in such models is measured in manyways and would include but not be limited to a reduction in mortalityrate, a reduction in bacterial cell counts determined by microscopicexamination of tissue or blood samples taken from the animals, or evenassessment of wound healing in the animals.

The following non-limiting examples are provided to further illustratethe present invention.

Example 1: Deinmmunization of Lysoataphin Catalytic Domain Materials andMethods

Reagents and Media.

Primers were ordered with standard desalting from IDT Technologies(Coralville, Iowa). PCR cleanup and gel extraction kits were from ZymoResearch (Irvine, Calif.). Commercial lysostaphin was purchased fromSigma (St. Louis, Mo.). Plasmid purification was performed using QIAPREPSpin Miniprep Kit (Qiagen; Valencia, Calif.). All enzymes were obtainedfrom New England BioLabs (Ipswich, Mass.), and all reagents from VWRScientific (Philadelphia, Pa.), unless otherwise noted. Peptides derivedfrom the lysostaphin catalytic domain were ordered from GenScript(Piscataway, N.J.), and were greater than 85% pure. MHC-II DR moleculeswere purchased from Benaroya Research Institute (Seattle, Wash.),anti-MHC-IIDR antibody from Biolegend (San Diego, Calif.), and DELFIAEu-labeled Streptavidin was from PerkinElmer (Boston, Mass.).

Epitope Prediction.

The T cell epitope content of the lysostaphin catalytic domain waspredicted using EpiMatrix, a scoring matrix whose predictions have beenshown to correlate well with the clinically observed immunogenicity oftherapeutics. EpiMatrix is a pocket profile method used to predict HLAbinding (Groot & Moise (2007) Curr. Opin. Drug Discover. Dev. 10). Inthis approach, a protein is divided into overlapping 9-mer peptides,each of which is then evaluated for its binding potential to HLAalleles. The eight most common HLA alleles (DRB1*0101, DRB1*0301,DRB1*0401, DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1301, DRB1*1501), whichare representative of more than 90% of the human population (Southwood,et al. (1998) J. Immunol. 160:3363-3373), were considered. Based onbinding potential, each peptide was assigned a correspondingstandardized Z score and then mapped onto the cluster immunogenicityscale, which represents the deviation in epitope content from what wouldbe expected for a randomly generated peptide (Groot, et al. (2013) Exp.Rev. Clin. Pharmacol. 6:651-662). Peptides scoring above 1.64 on theEpiMatrix “Z” scale (approximately the top 5%) were considered to belikely to bind to the corresponding MHC molecule, while peptides scoringin the top 1% (above 2.32 on the scale) were extremely likely to bind(Koren, et al. (2007) Clin. Immunol. 124:26-32). If a peptide had ascore higher than 1.64 for four or more alleles, it was said to containan EpiBar (Weber, et al. (2009) Adv. Drug Deliv. Rev. 61:965-976).

Homology Modeling of Lysostaphin.

EpiSweep analyzes deimmunized variants based on two quantified measures:epitope score and force field energy value. However, the algorithm usesthe structure of the protein to calculate energy. Since the crystalstructure of lysostaphin was not known, a lysostaphin model wasconstructed based on the available crystal structures of proteinssimilar to the catalytic domain of lysostaphin. The templates wereselected by comparing the sequence of lysostaphin to the PDB databaseand selecting three highly similar protein structures (PDB accessioncode: 2BOPA, 2B44A, and 1QWYA). For homology modeling purposes, sequenceidentity above 30% is considered sufficiently accurate (Rost (1999)Prot. Eng. Design Select. 12:85-94). All of the template structuresbelong to LytM, an autolysin from S. aureus, which has 48% sequenceidentity and 63% similarity to the catalytic domain of lysostaphin (Lu,et al. (2013) supra).

The catalytic domain models were built using LytM crystal structures byemploying MODELLER, a homology modeling protocol that builds athree-dimensional structure of proteins based on coordinates of templatestructures (Shen & Sali (2006) Protein Sci. 15:2507-2524). Twohundred-fifty models were generated and the most accurate one wasselected in terms of the DOPE statistical potential (Shen & Sali (2006)supra).

For regions of the protein for which there was no sufficient coordinateinformation to allow complete and accurate construction of the model, aprotein loop modeling method, FREAD, was used to remodel the existinggaps (Choi & Deane (2010) Proteins 78:1431-1440). The obtained homologymodel was minimized against AMBER99sb with an implicit solvent model(GB/SA). The aglycosylated wild-type (125 NPT) was modeled by applyingin silico mutations to the original model using Scwrl4 (Krivov, et al.(2009) Proteins: Struct. Funct. Bioinform. 77:778-795), followed byenergy re-minimization.

Evolutionary Information.

The process of deimmunization requires mutation of residues that arepredicted to contribute to MHC binding. However, T cell epitopes can bepresent in any part of the protein. Thus, random selection of mutationscould lead to disruption of proper folding and function. This problemcan be mitigated by adopting point mutations found in sequences remotelysimilar to the target sequence. To determine which mutations could beused in the deimmunization process, a total of 10,000 homologs toLST^(CAT) were collected by running PSI-BLAST (3 iterations, e-value<0.001). These sequences were filtered to remove those with >50% gaps or<35% sequence identity to the wild-type. A diverse set of 218representative sequences was subselected so as to have at most 90%sequence identity to each other. Allowed mutations were those predictedto delete at least one putative epitope while appearing as frequently asexpected in terms of a background probability distribution (McCaldon &Argos (1988) Proteins 4:99-122). Additional filters excluded mutationsto/from Pro and Cys, mutations involving active site residues (32His,36Asp, 82His, 113His and 115His), and mutations previously found to bedetrimental (Thr43Asp, Ser50Asp, Asn121Asp, and Leu135Ser).

EpiSweep.

Structure-based EpiSweep is a protein redesign tool that allows forprotein deimmunization while retaining protein stability andfunctionality. The algorithm combines validated immunoinformatics andstructural modeling to produce Pareto optimal designs that can then beselected experimentally for the best immunogenicity, stability, andactivity scores. The method by which EpiSweep selects the optimaldesigns has been described (Parker, et al. (2013) J. Computation. Biol.20:152-165). Briefly, the algorithm addresses the stability concern byassuming the protein backbone as rigid and selecting the best side-chainconformations from a discrete set of rotamers, which are chosen tominimize total protein energy. All rotamers and rotamer pairs areevaluated for potential clashes with the backbone and with each other.For example, conformations found to contain rotamers with a significantvan der Waals radii overlap or with exceptionally high intra- orinter-rotamer energies are discarded (Parker, et al. (2013) supra).

For EpiSweep analysis of the lysostaphin catalytic domain, themutational load was allowed to vary from two to eight mutations and thealgorithm was constrained to disallow mutations at the active site(His32, Asp36, His82, His113, and His115). Furthermore, the algorithmwas to generate not only Pareto optimal plans at each mutational load(designs with the lowest possible rotamer energies), but also theadditional 19 suboptimal plans (designs that have successively worserotamer energies as compared to the Pareto optimal plans). This analysiswas performed to correct for any possible mistakes that may arise due tothe rigid backbone assumption, since proteins are characterized by ahigh degree of flexibility. In fact, it has been observed that furtherside chain optimization changes Pareto optimality of EpiSweep designs,and thus may affect design selection (Parker, et al. (2013) supra).

EpiSweep Analysis of Ser126Pro Backbone.

For analysis using the Ser126Pro lysostaphin backbone, the algorithmconsidered only the 12 well-tolerated mutations (Ser124Gly, Ser122Asp,Asn121Gly, Arg118Thr, Ile99Gln, Lys95Glu, Tyr93His, Leu83Met, Lys46His,Ile41Glu, Asn13His, and Asn12Gly). Since Ser122Asp mutation was anextremely efficient epitope remover (Table 6), the algorithm was alsoconstrained to include this mutation in all generated plans. Additionalpost-processing energy minimization of the EpiSweep designs wasperformed using molecular modeling software TINKER against AMBER(AMBER99sb) force field and an implicit solvent model (GB/SA).

Plasmids and Strains.

P. pastoris strain GS115 and expression vector pPIC9 were obtained fromInvitrogen (Grand Island, N.Y.). S. aureus strain SA113 and S. aureussubsp. aureus (ATCC 25923) were obtained from the American Type CultureCollection (Manassas, Va.). Other strains of S. aureus (methicillinsensitive strains 6445 and 3425-1, and MRSA strain 3425-3) were clinicalisolates.

Synthesis of Lysostaphin Gene Optimized for P. pastoris Expression.

Synthesis of a synthetic lysostaphin gene was performed as described(Zhao, et al. (2014) Appl. Environ. Microbiol. 80:2746-53). The majorityof the codons was replaced to reflect the codon preference by P.pastoris (Zhao, et al. (2000) Sheng Wu Gong Cheng Xue Bao 16:308-11). Todisrupt long A+T nucleotide stretches in the gene sequence, second-mostfrequent codons were introduced as needed.

PCR-Based Synthesis of Single Point Mutants.

Lysostaphin single point mutants were synthesized as described (Zhao, etal. (2014) supra). Briefly, the mutations were introduced using spliceoverlap extension PCR with primers listed in Table 1. For instance, theSer122Gly mutation was introduced by first amplifying lysostaphin geneusing Syn_F and S122G_R, and S122G_F and Syn_R primers. The resulting(gel-purified) gene fragments were then mixed at an equimolar ratio, andused as a template in a subsequent reaction using Syn_F and Syn_Rprimers. The final product was the full-length lysostaphin gene with theSer122Gly mutation. All PCR reactions were performed using PHUSIONHigh-Fidelity DNA polymerase. The lysostaphin gene harboring the desiredmutation was then digested with EcoRI and XhoI, and ligated into thepPIC9 plasmid using T4 DNA ligase. The end product of ligation was thelysostaphin gene fused to the alpha mating factor secretion signal fromSaccharomyces cerevisiae. The resulting plasmid was transformed into E.coli DH5a electrocompetent cells (F⁻□80lacZΔM15Δ (lacZYA-argF) U169recA1 endA1 hsdR17 (r⁻ _(K) m⁺ _(K)) phoA supE44 λ⁻thi1 gyrA96 relA1).Clones were evaluated for the presence of lysostaphin gene using Syn_Fand Syn_R primers and sequenced to confirm the presence of mutations(primers AOX1_F and AOX1_R).

TABLE 1 SEQ ID Primer Sequence (5′→3′) NO: N120_RACCCTTCTTGTAGTTACCCAACCATTGAGCGGA 50 N12G_FTCCGCTCAATGGTTGGGTAACTACAAGAAGGGT 51 N13H_R CGATGAATTCTTACTTGATGGTACCCCA52 N13H_F ATCGCTCGAGAAAAGAGCTGCTACCCA 53 CGAGCACTCCGCTCAATGGTTGAACCACTACF38GR GGTACCGATGTTCATACCGAAGTCAACACCGTA 54 F38G_FTACGGTGTTGACTTCGGTATGAACATCGGTACC 55 I41E_RAGCCTTGACTGGGGTACCCTCGTTCATGAAGAA 56 I41E_FTTCTTCATGAACGAGGGTACCCCAGTCAAGGCT 57 K46H_RACCGGAGGAGATAGCGTGGACTGGGGTACCGAT 58 K46H_FATCGGTACCCCAGTCCACGCTATCTCCTCCGGT 59 L83M_R TACTTGGACATGTGCATGTACCATTG60 L83M_F CAATGGTACATGCACATGTCCAAGTA 61 Y93H_RTTGACCAGCCTTGACGTGGTCACCGACCTTGAC 62 K95E_RGATGATTTGACCAGCCTCGACGTAGTCACCGAC 63 K95E_FGTCGGTGACTACGTCGAGGCTGGTCAAATCATC 64 Y93H_FGTCAAGGTCGGTGACCACGTCAAGGCTGGTCAA 65 I99Q_RACCGGACCAACCGATTTGTTGACCAGCCTTGAC 66 I99Q_FGTCAAGGCTGGTCAACAAATCGGTTGGTCCGGT 67 R118T_RGAAGGAGTTGACCATGGTTTGGAAGTGCAAGTG 68 R118T_FCACTTGCACTTCCAAACCATGGTCAACTCCTTC 69 N121G_RTGGGTTGGAGAAGGAACCGACCATTCTTTGGAA 70 N121G_FTTCCAAAGAATGGTCGGTTCCTTCTCCAACCCA 71 S122D_RGGTTGGGTTGGAGAAGTCGTTGACCATTCTTTG 72 S122D_FCAAAGAATGGTCAACGACTTCTCCAACCCAACC 73 S122G_RGGTTGGGTTGGAGAAACCGTTGACCATTCTTTG 74 S122G_FCAAAGAATGGTCAACGGTTTCTCCAACCCAACC 75 S124G_RTTGAGCGGTTGGGTTACCGAAGGAGTTGACCAT 76 S124G_FATGGTCAACTCCTTCGGTAACCCAACCGCTCAA 77 S124Y_RTTGAGCGGTTGGGTTGTAGAAGGAGTTGACCAT 78 S124Y_FATGGTCAACTCCTTCTACAACCCAACCGCTCAA 79 Syn_R CGATGAATTCTTACTTGATGGTACCCCA80 Syn_F ATCGCTCGAGAAAAGAGCTGCTACCCAC 81 AOX1_R GCAAATGGCATTCTGACATCC 82AOX1_F GACTGGTTCCAATTGACAAGC 83 AflII_RAACGTAACCAGCGGACTTAAGGAATGGCATTGGGTC 84 AflII_FGACCCAATGCCATTCCTTAAGTCCGCTGGTTACGGT 85 T118R_RGAAGTCGTTGACCATTCTTTGGAAGTGCAAGTG 86 T118R_FCACTTGCACTTCCAAAGAATGGTCAACGACTTC 87

Cloning of LST Integrated Designs.

Lysostaphin variants that were not expressing in the first round ofscreening and contained the Arg118Thr mutation were synthesized withoutthat mutation and re-examined for expression. Splice overlap extensionPCR was used to revert the Arg118Thr mutation back to wild-type (Thr118)with primers T118R_F and T118R_R (Table 1).

To prepare the pPIC9 plasmid for insertion of synthesized genes, asilent mutation was introduced into the lysostaphin linker (residues135-136) to accommodate a cutting site for the restriction enzyme AflII.To introduce the necessary mutations, splice overlap extension PCR wasused with primers AflII_F and AflII_R (Table 1). The synthetic geneswere digested with XhoI and AflII restriction enzymes, and ligated insimilarly digested pPIC9 plasmid using T4 DNA ligase. The resultingplasmid was transformed into E. coli DH5a electrocompetent cells and theresulting clones were sequenced to confirm the presence of mutations.

P. pastoris Expression and Purification.

After DH5a clones were confirmed for the presence of correct catalyticdomain mutation by sequence analysis, the purified plasmid was digestedwith SacI High-Fidelity restriction enzyme prior to electroporation intoP. pastoris strain GS115. The resulting transformants were grown on MDplates (1.34% yeast nitrogen base, 0.000004% biotin, 2% dextrose and 1%agar). For expression studies, clones were grown in BMGY media (1% yeastextract, 2% peptone, 1.34% yeast nitrogen base, 0.000004% biotin, 1%glycerol, 100 mM phosphate buffer, pH 6) at 30° C. in 500 ml shakeflasks covered with four layers of cheese cloth for enhanced oxygen flowto the yeast. After 24 hours, cells were centrifuged at 3,000 rpm in atable top centrifuge for 10 minutes. The cells were then resuspended in100 ml of BMMY induction media (1% yeast extract, 2% peptone, 1.34%yeast nitrogen base, 0.000004% biotin, 0.5% methanol, 100 mM phosphatebuffer, pH 6) and allowed to grow for the next 48 hours at 30° C. At12-hour intervals, 100% methanol was added for a final concentration of1%. After 48 hours of induction, shake flask culture was centrifuged ina table top centrifuge at 3,000 rpm for 15 minutes. The resultingsupernatant was filtered to remove any yeast cells and diluted 1:5 with10 mM KH₂PO₄ buffer at pH 7.5. Diluted supernatant was flowed over agravity column packed with 500 μl SP-SEPHAROSE Fast Flow resin (GEHealthcare; Cleveland, Ohio). The column was washed with 5 ml of 50 mMNaCl in 10 mM KH₂PO₄ at pH 7.5. Protein was eluted with 500 μl aliquotsof 200 mM NaCl in 10 mM KH₂PO₄, pH 7.5. The purity of lysostaphin wasdetermined using SDS-PAGE. The protein concentration was quantifiedusing ND-1000 Spectrophotometer (NanoDrop Technologies; Wilmington,Del.). To ensure accuracy, protein absorbance measure was adjusted usingthe absorbance adjustment factor of 0.4 for both wild-type lysostaphinand its variants. Briefly, the adjustment factor was calculated as theinverse of the reported Abs 0.1% (=1 g/L) value (ProtParam, ExPASy).

Lytic Assay of Culture Supernatant.

S. aureus cells were grown either to mid-log or to saturation in trypticsoy broth (TSB) at 37° C. with shaking. The cells were harvested bycentrifugation and washed once in phosphate buffered saline (PBS: 2.7 mMKCl, 1.5 mM KH₂PO₄, 8.9 mM Na₂HPO₄, 136.9 mM NaCl, pH 7.4). The assaywas performed in 96-well, black, clear bottom plates from GreinerBio-One (Monroe, N.C.). The final (250 μl) reaction was composed of 10μl of P. pastoris culture supernatant, S. aureus cells at OD600=1.5, and5 μM SYTOX Green (Thermo Fisher Scientific; Waltham, Mass.), all in PBS.Data were collected using SPECTRAMAX GEMINI Fluorescence MicroplateReader (Molecular Devices; Sunnyvale, Calif.) using anexcitation/emission of 504/523 nm, and rates were determined from theslope of the steepest linear portion of the trace. Each assay wasperformed with 500 ng of commercially-sourced lysostaphin (ssLys) as aninternal control.

The amount of protein used in each assay was estimated from SDS-PAGEgels. Briefly, 10 μl of culture supernatant was run on a SDS-PAGE geltogether with standards of 0.5 μg, 0.7 μg, and 1 μg of ssLys in separatelanes. The gel was stained using GELCODE Blue Stain Reagent from ThermoFisher Scientific (Carlsbad, Calif.) and bands quantified using QuantityTools from Image Lab 5.1 software (Bio-Rad Laboratories; Hercules,Calif.).

Lytic Assay Using Purified Protein.

The activity of purified lysostaphin variants was examined using theSYTOX kinetic assay as previously described in Chapter 2, Section 3.2.6.200 ng of purified enzyme (instead of culture supernatant) was used ineach reaction.

MIC Assay.

The MICs of wild-type lysostaphin and its variants were determined byadding 2-fold serial dilutions of enzymes into wells of a polypropylene96-well plate (Costar 3879) containing ˜40,000 S. aureus SA113 (or S.aureus 6445, 3425-1, 3425-3) cells in Muller Hinton broth (BD)supplemented with 2% NaCl and 0.1% bovine serum albumin, yielding atotal volume of 100 μl. Plates were grown overnight at 37° C. withshaking at 900 rpm on an Orbit P4 orbital shaker (Labnet; Edison, N.J.).The inhibitory activity of purified lysostaphin was determined by theconcentration of enzyme that completely inhibited bacterial growth. Theassay was performed in triplicate for each enzyme.

PNGase F Treatment.

P. pastoris culture supernatant was treated with 1 μl of 10× G7 bufferand the same volume of REMOVE-IT PNGase F. The reaction was incubatedfor 1 hour at 37° C. and the results analyzed by SDS-PAGE.

Saturation Mutagenesis.

Saturation mutagenesis at position 125 of the lysostaphin catalyticdomain was carried out using known methods (Zhao, et al. (2014) supra).Briefly, saturation mutagenesis was performed by splice overlapextension PCR using the lysostaphin synthetic gene as a template anddegenerate NNK primers. The resulting 32-member library was transformedinto P. pastoris, and transformants were grown on YPD medium (1% yeastextract, 2% peptone, 1% methanol, 1% agar) at 30° C. for 48 hours. Tofind yeast clones expressing the active enzymes, molten top agar (0.5%yeast extract, 1% peptone, 1% NaCl, 0.75% agar) containing S. aureusSA113 cells was poured over YPM yeast plates and incubated at 37° C. for10 hours. Halo-forming colonies were picked out and amplified usingprimers Syn_F and Syn_R, and the genes were sequenced using primersAOX1_F and AOX1_R.

MIC Assay Using P. pastoris Culture Supernatant.

Lysostaphin MIC was determined essentially as described (Zhao, et al.(2014) supra). Briefly, 100 μl aliquots of P. pastoris culturesupernatant were serially diluted in TSB. Each well was inoculated with100 μl of ˜10⁶ CFU/ml S. aureus SA113 in TSB. Microplates were incubatedat 37° C. for 24 hours. The inhibitory activity in culture supernatantswas assessed as the MIC₅₀, the treatment dilution yielding 50%inhibition of growth. MIC₅₀ was quantified by measuring light scatteringat 650 nm in a microplate reader.

Thermostability.

The relative thermostability of the lysostaphin variants was determinedby differential scanning fluorimetry, as previously described (Niesen,et al. (2007) Nat. Protocols 2:2212-2221). Proteins and SYPRO Orangewere diluted in PBS (final concentrations of 100 μg/ml and 5× in 20 μlreaction volume, respectively), and fluorescence was quantified at1-degree increments from 25 to 94° C. using an Applied Biosystems ABI7500 fast real-time PCR system. The reactions were performed using PCRPlates for Fast Thermocyclers (VWR; Radnor, Pa.). Fluorescence wasquantified using the preset TAMRA parameters. Melting temperatures weredetermined by data analysis with the ‘DSF Analysis v3.0.xlsx’ EXCELsheet and GraphPad Prism v.6.02 software.

MHC Binding Assays. MHC II competition binding assays were performedusing a 384-well high throughput assay as previously described (Salvat,et al. (2014) supra). Binding assays were performed for the eightalleles: DRB1*0101, 0301, 0401, 0701, 0801, 1101, 1301, and 1501.Briefly, 100 nM biotinylated control peptides composed of known peptideantigens for each MHC II allele were incubated in polypropylene 384-wellplates with 50 nM purified recombinant MHC II protein and serialdilutions of LST or variant peptide fragments (100 μM to 10 nM).Peptide-MHC II complexes were captured from equilibrated solutions usingthe conformation specific anti-HLA-DR antibody L243 coated on highbinding ELISA plates. Bound control peptide was quantified using theDELFIA streptavidin-Europium conjugate and time resolved fluorescence(SpectraMax Gemini Fluorescence Microplate Reader).

Biofilm Degradation Assay.

S. aureus SA113 was grown overnight in TSB at 37° C. shaking. The cellswere then diluted 1:100 in TSB supplemented with 5% ethanol and 0.1%glucose and 100 μl of cell suspension was added to wells of a 96-wellplate (Costar 3595). The cells were left to form biofilms overnight at37° C. without shaking. The resulting biofilms were then washed threetimes in water and treated with 200 ng of enzyme in 100 μl for 75minutes. No-treatment wells contained PBS with 0.1% BSA. The plates werewashed three times in water after the treatment and stained with 0.1%crystal violet for 15 minutes. The plates were then washed again threetimes in water and allowed to dry. Two hundred μl of 30% acetic acid wasadded to each well and allowed to dissolve the crystal violet stain for15 minutes at 25° C. with shaking. Destain (150 μl) was transferred to anew 96-well plate and the absorbance of each well was measured in aSPECTRAMAX 190 spectrophotometer (Molecular Devices; Sunnyvale, Calif.)at 550 nm.

Murine Lung Infection Model.

Overnight LB cultures of S. aureus strain ATCC 25923 were pelleted,washed twice with PBS, and resuspended to give 10⁸-10⁹ colony formingunits (CFU) in 40 μl of PBS. The actual inoculum was determined byserial dilution of the input bacterial suspension on LB agar (DIFCO),followed by incubation at 37° C. for 24 hours. Adult female C57BL/6Jmice (age, 8 to 12 weeks; Jackson Laboratories, Detroit, Mich.) wereanesthetized briefly with isoflurane and inoculated with 40 μl ofbacterial suspension via oropharyngeal aspiration. At 1 hourpost-infection, a second 40 μl PBS inoculation containing either 2.5 μgwild-type LST, 2.5 μg variant Flex 5, 2.5 μg variant Flex 9, or a blankcontrol. At 24 hours post-infection, mice were sacrificed and lungs wereexcised, placed into 1 ml of cold PBS, and homogenized. Viable bacterialcounts in the lung homogenate were determined by plating serialdilutions onto LB agar, followed by incubation at 37° C. for 24 hours.

HUMI Murine Immunogenicity Studies.

HUMI mice were constructed by surgical transplantation of human bonemarrow, liver, and thymus tissues into NOD/SCID/γ_(c) ^(−/−) mice(Dartmouth Transgenics & Genetic Constructs Shared Resource) asdescribed (Brainard, et al. (2009) J. Virol. 83:7305-21). All animalswere humanized from the same human donor. Mice used experimentally hadhuman lymphocytes as a minimum of 25% of their total peripheral bloodleukocytes. Fourteen weeks post-engraftment, 12 female HUMI mice weredivided into 3 groups of 4 each and immunized with a single 50 μlsubcutaneous injection of 100 μg wild-type LST, 100 μg variant Flex 5,or 100 μg variant Flex 9 in complete Freund's adjuvant (CFA). Two weeksfollowing the immunization, mice were sacrificed and splenocytes wereharvested and pooled for each group. Pooled splenocytes (5×10⁵/well)were plated in triplicate into 96-well plates with medium containing 5%fetal calf serum, 1-glutamine, antibiotics, and a final concentration of10 μg/ml LST or variants (or 1% DMSO as a control). After 72 hours ofincubation, wells were pulsed with 1 μCi of [³H]thymidine (Dupont NEN,Boston, Mass.) and harvested 6 hours later onto UNIFILTER 96-well GF/Cplates for assessment of thymidine incorporation by scintillationcounting (Packard MicroSant NXT counter).

Transgenic DR4 Murine Immunogenicity Studies.

Twelve female 6-8 week old DR4 transgenic mice (Abb Knockout/TransgenicHLA-DR4;B6.129S2-H2-Ab1tm1GruTg(HLA-DRA/H2-Ea,HLA-DRB1*0401/H2-Eb)1Kito; TaconicFarms, Germantown, N.Y.) were divided into four groups of three each andimmunized with 50 μl subcutaneous injections of wild-type LST using oneof the following four schemes: (i) initial immunization with 100 μgenzyme in CFA, followed by 100 μg boosts in incomplete Freund's adjuvant(IFA) on days 14 and 28; (ii) initial immunization with 20 μg enzyme inCFA, followed by 20 μg boosts in IFA on days 14 and 28; (iii) initialimmunization with 100 μg enzyme in PBS buffer, followed by 100 μg boostsin PBS buffer on days 7, 14, 21 and 28; (iv) initial immunization with20 μg enzyme in PBS buffer, followed by 20 μg boosts in PBS buffer ondays 7, 14, 21 and 28. Serum IgG antibody titers against wild-type LSTwere measured on days 13, 20, 27, 34, and 62. Five weeks after the finalboost, all 12 mice exhibited equivalent maximum ELISA signals at a 1:40serum dilution and all signals were within 20% at a 1:160 dilution. Micewere housed without further manipulation until week 23 of the study, atwhich time serum IgG antibody titers were again measured and mice weredivided into two experimental arms having equivalent average antibodytiters. Note that during the week 9 to week 23 recovery period, two mice(the lowest titer 100 μg no adjuvant and one of the high titer 100 μgadjuvant) began suffering hair loss, weight loss, and reduced mobilityand were sacrificed as per the IACUC approved protocol. At week 24, onearm was rechallenged with 100 μg wild-type LST in IFA and the other armwith 100 μg variant Flex 5 in IFC. At week 26, mice were sacrificed andsplenocytes were harvested and pooled for each group. Proliferationassays were conducted as described above.

Bioinformatics Analysis.

Sequence alignment of lysostaphin and its homologous sequences ALE-1 andLytM was performed using ClustalW.

Epitope Prediction

EpiMatrix analysis of the lysostaphin catalytic domain of an Asn125Glnmutant (Zhao, et al. (2014) supra) showed that the domain had manypredicted T cell epitopes, with a total epitope score of 46. The proteinhad 14 instances of predicted top 1% binders (score>2.32), and 32instances of top 5% binders (score>1.64). The sequence was also found tocontain three EpiBars (peptides which have a score of 1.64 or higher fora minimum of four alleles), with the peptide ¹¹⁶FQRMVNSFS¹²⁴ (SEQ IDNO:88) predicted as highly immunogenic for all eight alleles (Table 2).

TABLE 2 SEQ Z Scores ID DRB1* DRB1* DRB1* DRB1* DRB1* DRB1* DRB1* DRB1*Peptide NO: 0101 0301 0401 0701 0801 1101 1301 1501 WLNNYKKGY 89 1.692.12 1.73 1.93 LNNYKKGYG 90 2.46 1.9 INGGMHYGV 91 1.93 VDFFMNIGT 92FFMNIGTPV 93 2.41 1.79 1.93 FMNIGTPVK 94 2.19 2.06 MNIGTPVKA 95 1.68IGTPVKAIS 96 1.85 VKAISSGKI 97 2.35 2.02 1.65 2.9 2.75 IENDGVHRQ 98 2.06VHRQWYMHL 99 1.99 WYMHLSKYN 100 2.72 YMHLSKYNV 101 1.86 2.29 2.72 2.01LSKYNVKVG 102 1.67 YVKAGQIIG 103 1.66 1.97 IIGWSGSTG 104 1.76 WSGSTGYST105 1.95 1.92 LHFQRMVNS 106 2.05 FQRMVNSFS 88 3.4 2.54 3.41 2.27 2.663.37 2.24 2.86 QRMVNSFSQ 107 1.71 1.8 MVNSFSQST 108 1.91 2.41 2.12

Selection of the Most Frequent Mutations for a Preliminary DesignAnalysis

EpiSweep yielded a total of 1,533 plans, 81 of which were evaluated asPareto optimal plans at mutational loads of two to eight mutations. Atthis point, it was possible to simply select a set of designs, whichwould then be subjected to experimental analysis for proper folding andactivity. However, previous efforts to produce deimmunized lysostaphinbased on sequence-based EpiSweep did not yield active variants.Structure-based EpiSweep was designed as an alternative method, whichwas supposed to produce better results. Given that inaccuratelysostaphin representation may have resulted in errors, an iterativefeedback strategy was used, wherein the 15 most frequent mutations(individually) were tested for their impact on protein folding(expression) and activity.

The results (Table 3) showed that the most frequent 15 mutations werepresent in 9-67% of total plans, and in 1-65% of Pareto optimal plans.The mutations were all found on the surface of the catalytic domain.Buried residues were not frequently used by EpiSweep. Indeed, buriedresidues Ser49Gly and Ser49Ala take positions as the 18^(th) and 20^(th)mutation, respectively. Amino acids that were predicted to contribute toMHC binding included basic (Arg/Lys), polar uncharged (Ser/Asn/Tyr), andnon-polar residues (Phe/Leu/Ile). These residues were replaced withnon-polar (Gly/Met), acidic (Asp/Glu), basic (His), or polar uncharged(Thr/Gln/Tyr) amino acids.

TABLE 3 Total Number Number of Surface/ of Plans with Optimal PlansBuried Mutation Mutation with Mutation Residue Arg118Thr 1031 (67%) 53(65%) Surface Ser124Gly 1012 (66%) 59 (73%) Surface Ser122Gly 954 (62%)57 (70%) Surface Lys95Glu 861 (56%) 50 (62%) Surface Phe38Gly 848 (55%)46 (57%) Surface Asn12Gly 604 (39%) 48 (59%) Surface Lys46His 516 (34%)25 (31%) Surface Tyr93His 516 (34%) 26 (32%) Surface Ser122Asp 348 (23%)18 (22%) Surface Leu83Met 336 (22%) 15 (19%) Surface Asn121Gly 262 (17%)11 (14%) Surface Ser124Tyr 246 (16%) 7 (9%) Surface Asn13His 179 (12%) 4(5%) Surface Ile41Glu 161 (11%) 10 (12%) Surface Ile99Gln 143 (9%) 1(1%) Surface Thr110Glu 136 (9%) 1 (1%) Surface Leu83Asn 136 (9%) 5 (6%)Surface Ser49Gly 117 (8%) 0 Buried Val120Gln 102 (7%) 3 (4%) SurfaceSer49Ala 69 (5%) 0 Buried

The mutations were predicted to significantly reduce the binding ofpeptides to the MHC and produce less immunogenic variants (Table 4). Inall generated peptides, it could be seen that the number of mutant hits(the number of alleles a peptide was predicted to bind) was lower thanthat of the wild-type peptides. For instance, the Arg to Thr mutation atposition 118 in the wild-type QRMVNSFSQ (SEQ ID NO:107) peptide waspredicted to delete both epitopes and resulted in a Z score of 0 acrossall the alleles. Some of the more immunogenic regions were harder totackle with a single mutation. Yet, it was observed that even a singlemutation could have a meaningful impact on reducing the totalimmunogenicity score. For instance, the Ser122Asp mutation deleted twoepitopes and reduced the overall hit number from 14 to 8.

TABLE 4 SEQ Z Scores ID DRB1*  DRB1* DRB1*  DRB1* DRB1*  DRB1* DRB1* DRB1* Mutation Peptide NO: 0101 0301 0401 0701 0801 1101 1301 1501 R118TQTMVNSFSQ 109 S124G QRMVNSFGQ 110 MVNSFGQST 111 1.83 1.77 S122GFQRMVNGFS 112 3.17 2.5 2.97 2.26 2.68 3.31 2.87 QRMVNGFSQ 113 MVNGFSQST114 2.01 K95E YVEAGQIIG 115 F38G VDFGMNIGT 116 FGMNIGTPV 117 2.1GMNIGTPVK 118 N12G WLGNYKKGY 119 2.02 LGNYKKGYG 120 1.98 K46H MNIGTPVHA121 IGTPVHAIS 122 VHAISSGKI 123 2.29 1.96 2.84 2.69 Y93H HVKAGQIIG 124S122D LHFQRMVND 125 FQRMVNDFS 126 3.28 1.93 3.02 1.75 2.98 1.97QRMVNDFSQ 127 MVNDFSQST 128 1.72 1.9 L83M VHRQWYMHM 129 MSKYNVKVG 130N121G QRMVGSFSQ 131 S124Y FQRMVNSFY 132 2.92 2.75 2.6 1.88 2.52 2.452.39 QRMVNSFYQ 133 MVNSFYQST 134 N13H WLNHYKKGY 135 2.21 I41E VDFFMNEGT136 FMNEGTPVK 137 2.16 MNEGTPVKA 138 EGTPVKAIS 139 I99Q YVKAGQQIG 1401.91 QIGWSGSTG 141 Mutations are shown in bold.Modification of Lysostaphin Sequence for P. pastoris Expression

Initial attempts to make S. simulans lysostaphin in P. pastoris werehampered by the lack of protein expression. Thus, the gene was modifiedfor expression in P. pastoris (Zhao, et al. (2014) supra). Briefly, thesequence of the wild-type lysostaphin was adjusted to reflect the codonpreference of P. pastoris. Additionally, a long segment withdisproportionate A+T content in the sequence was identified anddisrupted. The new version of the gene (SYN lysostaphin) was found toyield up to 80 mg/L of protein in shake flask culture, and 500 mg/L in a2 L bioreactor (Zhao, et al. (2014) supra).

Subsequent expression experiments conducted with SYN lysostaphin showedthat the protein migrated as a doublet in SDS-PAGE. It was suspectedthat the observed doublets were due to protein N-glycosylation. Closeexamination of the lysostaphin sequence revealed that it contained aglycosylation sequon at position 125. The presence of the N-glycan wasconfirmed by PNGase treatment of the culture supernatant. Once treated,the protein migrated as a singlet in SDS-PAGE.

An attempt to disrupt the Asn125 glycosylation sequon was made byintroducing conservative Asn->Gln, Asn->Ser, and Asn->Asp single pointmutations. The results of this analysis indicated that these mutantsexhibited similar expression levels but had 10-, 20- and 40-fold loweractivity than the wild-type enzyme, respectively.

In subsequent studies, a library was constructed by saturationmutagenesis to determine which other residues besides Asn could betolerated at position 125 and still allow for disruption ofN-glycosylation sequence (Zhao, et al. (2014) supra). The libraryresults, and subsequent alignment of lysostaphin sequence with itshomologues (ALE-1 and LytM), showed that Asn125 is a conserved residue,such that focus needed to be placed on mutating other residues of theglycosylation sequon. Thus, two other mutants, Ser126Pro and Thr127Ala,were synthesized and found to successfully prevent N-glycosylation. Themutants were compared to the wild-type lysostaphin and found to exhibitequivalent activity. Since a fully active aglycosylated mutant wasdesired, the lysostaphin Ser126Pro was selected for further experimentalevaluation with the EpiSweep algorithm.

EpiSweep Correction for Aglycosylated Lysostaphin

The Ser126Pro lysostaphin backbone was analyzed with EpiMatrix andEpiSweep to determine the epitope score and compare the most frequentmutations. The results showed that switching to the Ser126Pro mutantincreased the predicted epitope score. Ser126Pro had a total epitopescore of 50, as compared to epitope score of 46 for the Asn125Glnbackbone (Table 2). EpiMatrix analysis also showed that Ser126Pro hadfive EpiBars (peptides which have a Z score of 1.64 or higher for aminimum of four alleles), while Asn125Gln had three. The Ser126Probackbone had 15 instances of predicted top 1% binders (score>2.32), and35 instances of top 5% MHC binders (score>1.64). Peptide ¹¹⁶FQRMVNSFS¹²⁴(SEQ ID NO:88) remained equally problematic, as in the Asn125Glnbackbone, and was predicted to be highly immunogenic for all eightalleles. The four new epitopes present in Ser126Pro backbone wereintroduced in peptides ¹¹⁹MVNSFSNPT¹²⁷ (SEQ ID NO:142) and¹²⁰VNSFSNPTA¹²⁸ (SEQ ID NO:143) (Table 5).

TABLE 5 SEQ Z Scores ID DRB1* DRB1* DRB1* DRB1* DRB1* DRB1* DRB1* DRB1*Peptide NO: 0101 0301 0401 0701 0801 1101 1301 1501 WLNNYKKGY 89 1.692.12 1.73 LNNYKKGYG 90 2.46 1.93 INGGMHYGV 91 1.9 VDFFMNIGT 92 1.93FFMNIGTPV 93 2.41 1.79 1.93 FMNIGTPVK 94 2.19 2.06 MNIGTPVKA 95 1.68IGTPVKAIS 96 1.85 VKAISSGKI 97 2.35 2.02 1.65 2.9 2.75 IENDGVHRQ 98 2.06VHRQWYMHL 99 1.99 WYMHLSKYN 100 2.72 YMHLSKYNV 101 1.86 2.29 2.72 2.01LSKYNVKVG 102 1.67 YVKACQIIG 103 1.66 1.97 IIGWSGSTG 104 1.76 WSGSTGYST105 1.95 1.92 LHFQRMVNS 106 2.05 FQRMVNSFS 88 3.4 2.54 3.41 2.27 2.663.37 2.24 2.86 MVNSFSNPT 142 2 1.9 2.79 1.78 2.25 VNSFSNPTA 143 2.031.96 2.05 2.55

EpiSweep analysis of the Ser126Pro backbone yielded a total of 2,333plans, 96 of which were evaluated as Pareto optimal plans at mutationalloads of two to eight mutations (compared with the Asn125Gln backbonewhich yielded a total of 1,533 plans, 81 of which were Pareto optimal).Table 6 compares the most frequent mutations found in the Asn125Gln andSer126Pro backbones. The mutation pattern was not significantly changedafter switching to the new backbone. Most mutations remained in the top15, with exception of only four mutations: Ser122Gly, Ser124Gly,Leu83Met and Ile99Gln moved to positions 16, 18, 23 and 24,respectively. The most frequent mutation, Arg118Thr, remained asdominant in both backbones. While in the Asn125Gln backbone all 15 mostfrequent mutations were surface exposed residues, the new backbonepushed a buried residue, Ser49Gly, to the top 15. Most mutations weresimilarly represented in total plans and optimal plans, with anexception of Ser124Gly, which changed from being present in 72% of theplans, to not being present in any of the optimal plans selected withthe Ser126Pro backbone. The most frequent 15 mutations (evaluated basedon the Asn125Gln backbone) were present in 2-82% of total plansgenerated using the Ser126Pro backbone, and in 0-88% of Pareto optimalplans.

TABLE 6 Percentage of Percentage of Plans with Optimal Plans RankingOrder Mutation Mutation with Mutation change Arg118Thr 67->82% 65->88%1->1 Ser124Gly  66->9%  72->0%  2->18 Ser122Gly 62->10%  70->4%  3->16Lys95Glu 56->53% 61->58% 4->2 Phe38Gly 55->18% 56->25%  5->11 Asn12Gly39->49% 59->78% 6->3 Lys46His 33->18% 30->18%  7->10 Tyr93His 33->45%32->58% 8->4 Ser122Asp 22->43% 22->54% 9->5 Leu83Met  21->4%  18->1%10->23 Asn121Gly 17->26% 13->32% 11->7  Ser124Tyr 16->12%  8->9% 12->15Asn13His 11->15%  4->10% 13->14 Ile41Glu 10->21% 12->26% 14->9  Ile99Gln 9->2%  1->3% 15->24 Thr110Glu  8->25%  1->27% 16->8  Leu83Asn  8->10% 6->8% 17->17 Ser49Gly  7->18%  0->27% 18->12 Val120Gln  6->6%  3->0%19->21 Ser49Ala  4->7%  0->0% 20->20

The 15 mutations based on the Asn125Gln backbone were still predicted tosignificantly reduce the binding of residues to MHC II and produce lessimmunogenic variants in the Ser126Pro backbone (Table 7). The predicteddeletions were largely similar to those obtained with Asn125Gln. Theoverall mutant hit rate with Asn125Gln was 35 (40 with Ser126Pro) andthe wild-type hit rate is 84 (88 with Ser126Pro), so the two backbonestargeted roughly the same number of epitopes.

The one significant difference observed was that the Arg118Thr mutation,which deleted two epitopes in the Asn125Gln backbone, did not delete anyepitopes in the Ser126Pro backbone. However, closer examination showedthat Arg118Thr helped other mutations delete more epitopes. Forinstance, when Arg118Thr was combined with Ser122Asp, the two mutationsdeleted a total of 13 epitopes. As such, the Arg118Thr mutation was noteliminated from further study. However, the Ser122Gly mutation wasexcluded since it targeted the same residue as Ser122Asp but onlydeleted three epitopes (as opposed to 10 that Ser122Asp was predicted toremove).

TABLE 7 SEQ Z Scores ID DRB1* DRB1* DRB1* DRB1* DRB1* DRB1* DRB1* DRB1*Mutation Peptide NO: 0101 0301 0401 0701 0801 1101 1301 1501 S124GMVNSFGQST 111 1.92 2.15 1.74 S122G FQRMVNGFS 112 3.17 2.5 2.97 2.26 2.683.31 2.87 MVNGFSQST 114 1.66 1.94 2.14 K95E YVEAGQIIG 115 F38G VDFGMNIGT116 FGMNIGTPV 117 2.1 GMNIGTPVK 118 N12G WLGNYKKGY 119 2.02 LGNYKKGYG120 1.98 K46H MNIGTPVHA 121 IGTPVHAIS 122 VHAISSGKI 123 2.29 1.96 2.842.69 Y931I HVKAGQIIG 124 S122D LHFQRMVND 125 FQRMVNDFS 126 3.28 1.933.02 1.75 2.98 1.97 MVNDFSNPT 144 1.86 2.26 VNDFSNPTA 145 L83M VHRQWYMHM129 MSKYNVKVG 130 N121G VGSFSNPTA 146 2.11 S124Y FQRMVNSFY 132 2.92 2.752.6 1.88 2.52 2.45 2.39 MVNSFYQST 134 2.02 N13H WLNHYKKGY 135 2.21 I41EVDFFMNEGT 136 FMNEGTPVK 137 2.16 MNEGTPVKA 138 EGTPVKAIS 139 I99QYVKAGQQIG 140 1.91 QIGWSGSTG 141 Mutations are shown in bold.

Expression Level and Activity of Lysostaphin Variants

Even though the algorithm was focused on deimmunization of only thecatalytic domain, it is important to note that the single point mutantswere tested in the context of a full-length, mature protein to ensureproper protein folding. Since lysostaphin is secreted into media, asimple analysis of lysostaphin activity from the culture supernatant wasconsidered a sufficient evaluation of the effect of single pointmutations on the protein.

The analysis of lysostaphin single point mutants revealed that allvariants were expressed in P. pastoris. Thus, none of the most frequentEpiSweep mutations abolished expression. The two mutations with thelowest expression levels were Arg118Thr and Ser124Tyr, with 47% and 35%of the wild-type expression level, respectively. Furthermore, 13 of thelysostaphin variants had activity that either equaled or exceeded thatof the wild-type enzyme. Point mutant Phe38Gly was one of the twomutations with activity lower than the wild-type (69%). The secondmutation with low activity was Ser124Tyr, with 16% of the wild-typeactivity. Since Phe38Gly and Ser124Tyr did not meet threshold activitylevels of >70% of the wild-type activity, neither enzyme was included infurther studies. While the expression level of the Arg118Thr mutationwas less than 50% of the wild-type enzyme, this enzyme was furtheranalyzed given the number of epitopes removed by this mutation.

Backbone Flexibility Adjustment

All designs obtained using EpiSweep assumed a rigid backbone (referredto herein as “rigid” backbone designs). However, since proteins areknown for their high level of flexibility, it was posited that keepingthe backbone fixed in the post-processing energy minimization step couldcompromise the accuracy of deimmunized variants by resulting inimprecise energy assessments. It has been observed that completely rigiddesigns differ drastically in energy-epitope score landscape from thedesigns in which the side chains were allowed to relax (while thebackbone was fixed) during minimization (Parker, et al. (2013) supra).Thus, to achieve flexibility in a computationally-permitting way,additional post-processing energy minimization of the EpiSweep designswas performed (referred to herein as “flexible” backbone designs). Theresults of this analysis showed that there was indeed a visible movementbetween the two backbones.

EpiSweep Analysis of Chosen Mutations

As described above, the expression and activity of the 15 most frequentmutations were analyzed. Based upon this analysis, two mutations weredropped due to unsatisfactory activity values. In addition, Ser122Glywas removed as it targeted the same residue as Ser122Asp, which deleted10 epitopes compared to the three epitopes of the Ser122Gly mutation.The most frequent mutation, Arg118Thr, did not delete any epitopes inthe context of the Ser126Pro backbone and had a relatively lowexpression level. However, Arg118Thr was maintained as it assisted othermutations in targeting epitopes. For instance, when combined withSer122Asp, the Arg118Thr mutation targeted a total of 13 epitopes. As aresult, the data set contained 12 different mutations: Ser124Gly,Ser122Asp, Asn121Gly, Arg118Thr, Ile99Gln, Lys95Glu, Tyr93His, Leu83Met,Lys46His, Ile41Glu, Asn13His, and Asn12Gly.

EpiSweep analysis was performed once again, but the algorithm wasconstrained to use only the 12 well-tolerated mutations. The mutationalload was allowed to vary from two to eight mutations and the algorithmwas forced not to introduce mutations at the active site (His32, Asp36,His82, His113, and His115). Since it was observed that the Ser122Aspmutation was an extremely efficient epitope remover, the algorithm wasconstrained to include this mutation in all generated plans. As before,the algorithm generated Pareto optimal plans and an additional 19suboptimal plans at each mutational load. The lowest epitope score wasfound to be 24, and could only be achieved with plans containing eightmutations. The highest epitope score was 40, and the plans with thisscore had only the forced Ser122Asp mutation.

A number of plans along the Pareto optimal curve (designs that have thelowest possible rotamer energy at a fixed epitope score and mutationalload) were selected. EpiSweep analysis yielded rigid backbone plans, anda total of 14 Pareto optimal plans at mutational loads ranging from twoto eight mutations were selected, with the epitope score in the range of24 (lowest) to 36. Since the wild-type lysostaphin backbone had anepitope score of 50, it was posited that this range covered designs thatwere significantly deimmunized.

To address the protein flexibility concern, 14 designs that were energyminimized post-processing (flexible backbone designs) were alsoincluded. The designs at the same epitope scores and mutational loads asthe rigid backbone designs were selected so that the two differentmethods of obtaining variants could be directly evaluated. For instance,if a Pareto optimal rigid plan with eight mutations and an epitope scoreof 24 were chosen, a flexible backbone design at the same mutationalload and epitope score was selected. When faced with several options,the flexible backbone design of the lowest rotamer energy was chosen.Because they were energy minimized, these designs did not appear on thePareto frontier. It should be noted that the two mutation plan wasshared between the rigid and flexible backbone designs.

Expression Level and Activity of Synthesized Variants

Each lysostaphin variant was grown in shake flask culture and examinedfor expression level by SDS-PAGE. The plans that were found to expressat a meaningful level in culture supernatants (by SDS-PAGE detection)were further characterized for their relative specific activities. Theresults showed that out of 28 rigid and flexible backbone plans, only 11were expressed. Expression levels of all variants except Rigid 1 werefound to be lower than that of the wild-type lysostaphin.

All of the plans (except Flex 1; SEQ ID NO:32) had activity lower thanthat of the wild-type enzyme, but only two had activity lower than thecommercial E. coli-produced lysostaphin. It was noted that there was asignificant difference between the activity of the wild-type lysostaphinproduced in P. pastoris and ssLys. Out of a total of 11 expressingplans, six were flexible backbone designs and five were rigid backbonedesigns. Rigid design mutational load ranged from two to six, andflexible from two to eight.

The activity level of the plans decreased as the number of mutationsincreased. This trend was better observed in flexible rather than therigid backbone designs. Similarly, in flexible plans, it was found thatthe expression level decreased with an increase in the mutational load.This trend was not observed in rigid backbone designs, as theirexpression level remained low regardless of the mutational load. Theonly rigid design that had a high expression level was the two mutationdesign Rigid 1, which was shared between the rigid and flexible designs(also referred to as Flex 14).

Analysis of Non-Expressing Mutants

Since less than a half of the synthesized designs expressed at ameaningful level, the non-expressing designs were analyzed for theirmutational patterns. This analysis showed that expressing andnon-expressing designs shared the majority of the mutations. Somemutations, such as Leu83Met, Tyr93His, and Lys95Glu, were present inmost of the non-expressing plans, but only in a few expressing plans.Similarly, Arg118Thr was present in only a few expressing plans but,unlike any other mutation (the forced Ser122Asp mutation excluded), itcould be found in all of the non-expressing plans (Table 8).

TABLE 8 No. Muta- Design ID N12G N13H I41E K46H L83M Y93H K95E I99QR118T N121G S122D S124G tions Wild-Type^(E) 0 Flex 1^(E) E Q D 3 Flex5^(E) H Q G D 4 Flex 6^(E) G E Q G D 5 Flex 8^(E) G E M Q G D 6 Flex9^(E) G E M E Q G D G 8 Flex 10^(E) G E G D 4 Flex 14^(E) E D 2 Rigid1^(E) E D 2 Rigid 2^(E) H T D 3 Rigid 3^(E) H H E T D 4 Rigid 13^(E) H HT D 4 Rigid 14^(E) H H H E T D 5 Flex 2^(N) E H T D G 4 Flex 3^(N) E E QT D 4 Flex 4^(N) E H E Q T D 5 Flex 7^(N) G E E Q T D G 6 Flex 11^(N) GE H E Q T D G 7 Flex 12^(N) H E E Q T D 5 Flex 13^(N) H E H E Q T D 6Rigid 4^(N) G E H M H E T D 7 Rigid 5^(N) G E M E Q T D G 7 Rigid 6^(N)G E M E T D 5 Rigid 7^(N) G H M T D 4 Rigid 8^(N) G E M H E T D 7 Rigid9^(N) G E T D 3 Rigid 10^(N) G E H E T D 5 Rigid 11^(N) H H M T D 4Rigid 12^(N) H H M H E T D 7 ^(E)Expressed. ^(N)Not expressed. Thedouble line divides expressing and non-expressing plans.

Since earlier results showed that mutation Arg118Thr had a relativelylow expression level (less than 50% of wild type), it was posited thatthe observed lack of expression in the majority of the synthesized plansmay have been due to the Arg118Thr mutation. To test this, the mutationwas reverted back to wild-type in all plans having this mutation.

Characterization of Both Original and Reverted Plans

All expressed plans were purified for further characterization ofactivity and stability. Furthermore, all of the designs that originallyhad the Arg118Thr mutation, but now had the wild-type residue at thesame position (reverted plans), were evaluated. The results of thisanalysis are shown in Table 9.

TABLE 9 % Wild- Flexible Rigid Type MIC Tm Design ID Energy EnergyEpiscore Activity (μg/mL) (° C.) Wild-Type −3929 −48.5 50 100 0.035159.2 Flex 1 −4130 −47.2 34 63 0.0833 55.4 Flex 5 −3954 −49.7 32 610.1042 55.7 Flex 6 −3978 −49.3 28 58 0.2083 54.1 Flex 8 −3974 −49.8 2660 0.2083 52.9 Flex 9 −4005 −50.8 24 57 0.1250 53.0 Flex 10 −3904 −49.430 62 0.0729 57.5 Flex 14 −4056 −47.4 36 69 0.0833 58.6 Rigid 1 −4056−47.4 36 69 0.0833 58.6 Rigid 2 −3784 −55.5 34 72 0.1042 49.2 Rigid 13−3699 −57.1 32 71 0.0521 46.9 Flex 2* −4016 −49.5 35 77 0.0833 58.4 Flex3* −4166 −47.7 33 61 0.2500 56.2 Flex 4* −4143 −50.7 33 36 0.2083 57.5Flex 7* −4084 −49.0 29 67 0.2500 54.7 Flex 11* −4059 −47.2 29 76 0.125052.8 Flex 12* −4079 −49.4 31 54 0.1250 54.8 Flex 13* −4056 −52.3 31 710.1667 54.2 Rigid 2* −3957 −49.5 37 80 0.1042 57.1 Rigid 3* −3884 −53.035 79 0.0833 57.3 Rigid 4* −3884 −55.3 26 55 0.2500 54.1 Rigid 5* −4080−55.5 27 66 0.1667 52.5 Rigid 6* −4013 −49.9 29 64 0.2083 53.3 Rigid 7*−3977 −49.4 31 80 0.0729 55.0 Rigid 9* −3980 −48.9 33 79 0.0729 56.5Rigid 10* −3993 −52.4 31 77 0.1458 55.6 Rigid 11* −3867 −51.7 33 700.0313 55.7 Rigid 13* −3871 −51.2 35 92 0.0625 56.9 Rigid 14* −3884−54.6 33 75 0.1042 56.1 Reverted plans are indicated with *.

Out of a total of 32 plans (original and reverted), 28 were found toexpress. The 28 that expressed well were purified and characterized fortheir activity and stability. During the preliminary analysis, Rigid 3and Rigid 14 were found to express weakly in the culture supernatant.Reverted versions of Rigid 8 and 12 did not significantly improveexpression. It is possible that these four plans had low expressionlevels due to the presence of other mutations besides Arg118Thr, but noobvious mutational patterns could be found. All other plans in which theArg118Thr mutation was changed back to the wild-type sequence were foundto express well. As such, the Arg118Thr mutation appeared to affectexpression for the majority of the plans.

The results showed that the variants had high levels of activity andstability, as compared to the wild-type enzyme. Activity values wereexpressed as % of the wild-type activity, and ranged from 92% (Rigid13*) to 36% (Flex 4*). The observed MIC values were close to thewild-type, with the highest being 0.25 μg/ml (Flex 3*, Flex 7* and Rigid4*). Tm values were also similar to that observed in the wild-typeenzyme, with the lowest Tm showing a decrease in stability of 12.3° C.(Rigid 13, Table 9).

This analysis indicated that an increase in the mutational load resultedin a decrease in activity/Tm values of the variants and an increase inMIC values. On average, rigid designs had a higher specific activity andlower MIC values than flexible designs. Flexible designs, however, had abetter overall stability than rigid designs, as evidenced by higheraverage Tm values. Based on the results of a two-tailed t-test, thedifferences observed between the two design groups were statisticallysignificant (p-values <0.03).

The designs were divided into the seven different groups (Table 10) forfurther characterization. Using the data from Table 9, the Pearsoncorrelation coefficients between the flexible/rigid energies, mutationalloads, activity, and stability for each design group were calculated.

TABLE 10 Pearson (P-value) Flexible Rigid Flexible Rigid All All AllOriginal Original Reverted Reverted Flexible Rigid Designs NM vs. −0.860.55 0.26 −0.80 −0.04 −0.61 −0.38 Act. (0.01) (0.63) (0.57) (0.00)(0.88) (0.02) (0.04) NM vs. 0.53 −0.59 −0.03 0.73 0.36 0.69 0.56 MIC(0.22) (0.59) (0.95) (0.01) (0.21) (0.01) (0.00) NM vs. −0.85 −0.94−0.85 −0.12 −0.79 −0.09 −0.24 Tm (0.02) (0.21) (0.02) (0.73) (0.00)(0.76) (0.22) FE vs. −0.36 0.77 0.72 0.20 0.21 0.08 0.39 Act. (0.43)(0.44) (0.07) (0.56) (0.47) (0.79) (0.04) FE vs. 0.22 −0.34 −0.79 −0.35−0.38 −0.34 −0.48 MIC (0.63) (0.78) (0.04) (0.29) (0.18) (0.23) (0.01)FE vs. −0.05 −1.00 −0.14 −0.02 −0.10 −0.59 −0.49 Tm (0.92) (0.03) (0.77)(0.95) (0.74) (0.03) (0.01) RE vs. 0.78 −0.8 0.25 0.52 0.3 0.36 −0.07Act. (0.04) (0.4) (0.59) (0.1) (0.3) (0.21) (0.75) RE vs. −0.4 0.27 0.05−0.4 −0.2 −0.2 0.05 MIC (0.35) (0.83) (0.91) (0.17) (0.56) (0.46) (0.79)RE vs. 0.63 1.00 −0.2 0.09 0.17 0.55 0.48 Tm (0.13) (0.02) (0.66) (0.8)(0.57) (0.04) (0.01) No. of 7 3 7 11 14 14 28 Designs NM, No. ofMutations; Act., Activity; FE, Flexible Energy; RE, Rigid Energy.

When considered together, all 28 designs showed a weak positivecorrelation between the number of mutations and the MIC value (Table 10,All Designs). Thus, as mutational load of the variants increased, theiractivity decreased (MIC value increased). A stronger positivecorrelation between the number of mutations and MIC was found in rigidreverted and all rigid plans.

Other correlations that were not obvious when all the plans wereanalyzed together become apparent when designs were evaluatedseparately. For instance, it was observed that a strong negativecorrelation existed between the mutational load and activity in theoriginal flexible plans, reverted rigid plans, and all rigid plans. Astrong negative correlation was also found between the number ofmutations and Tm values in flexible original, flexible reverted, and allflexible plans.

Overall, strong correlations were not observed between energy andactivity/stability terms when all designs were considered together. Theonly meaningful correlation observed was a weak negative correlationbetween the flexible energy and Tm (Table 10, All Designs). Thecorrelations observed between the flexible energy and activity terms hadopposite signs than what was expected. On the other hand, correlationsbetween the rigid energy and activity were not significant, while thecorrelation between rigid energy and Tm had incorrect sign. Thus, tofurther examine whether energy was a reliable predictive tool, thecorrelations between energy components and experimentally determinedactivity and stability values were evaluated.

Even though flexible and rigid energies were not great predictors ofactivity, it was found that energy due to solvent interactions could beused instead (Table 11). As expected for a functional predictor, solventenergy had a negative (although weak) correlation with activity, and astronger positive correlation with MIC values.

TABLE 11 Pearson Correlation Coefficient Activity MIC Tm Flexible Energy0.391 −0.479 −0.0488 Energy Due to 0.402 −0.0514 −0.0282 IntermolecularForces Energy Due to 0.449 −0.605 −0.291 Charge-Charge InteractionsEnergy Due to −0.397 0.556 0.064 Solvent Interactions Rigid Energy−0.065 0.054 0.484

Taken together, these results showed that flexible backbone energy was arelatively good predictive tool of stability, while energy due tosolvent interactions seemed to be the best predictor for experimentalactivity observed using a MIC assay.

Characterizing Immunoreactivity of Lysostaphin Variants

The predicted epitopes in the wild-type lysostaphin catalytic domainwere broadly distributed throughout the lysostaphin sequence, but themajority of the epitopes could be grouped into five clusters (FIG. 2).Most epitopes were predicted to have a high number of binding events.

To evaluate the relative immunogenicity of the variants, a total of 26synthetic peptides spanning the sequence of the lysostaphin catalyticdomain were designed. In particular, focus was placed on the fiveimmunogenic clusters shown in FIG. 2. For each cluster listed, awild-type peptide and the corresponding mutated peptide were designed.The mutated peptides contained both the single mutation and mutationalcombination that appeared in plans.

In the context of synthetic peptides, each mutation was evaluated forits predicted potential to eliminate epitopes. EpiBars (peptides thathad a Z score of 1.64 or higher for a minimum of four alleles on theEpiMatrix immunogenicity scale) were found in cluster 2 (⁴⁵VKAISSGKI⁵³,SEQ ID NO:97), cluster 3 (⁸⁰WMHLSKYNV⁸⁸, SEQ ID NO:147), and cluster 5(¹¹⁶FQRMVNSFS¹²⁴, SEQ ID NO:88; ¹¹⁹MVNSFSNPT¹²⁷, SEQ ID NO:142; and¹²⁰VNSFSNPTA¹²⁸, SEQ ID NO:143).

Each peptide from was experimentally evaluated for its binding potentialto eight MHC II alleles in a high-throughput MHC II binding assay. TheEpiSweep-generated mutations generally lessened the binding of thepeptides to the MHC. Out of the 168 pair-wise comparisons, mutationsdecreased binding affinity in 73 cases, had no effect in 60 cases andincreased binding in 35 cases. A peptide was classified as a strongbinder if an IC₅₀ value of less than 0.1 μM was observed, moderate if anIC₅₀ value was in the 0.1-1 μM range, and weak if an IC₅₀ value was inthe 1-10 μM range. All peptides above 10 μM were considered non-binders.

Using the 10 μM cutoff to separate binders from non-binders, EpiMatrixpredictions (at 5% threshold) were compared with the MHC II bindingresults. The percentage of true positives (correctly predicted binders),true negatives (correctly predicted non-binders), false positives(incorrectly predicted binders), and false negatives (incorrectlypredicted non-binders) were calculated. The results showed that theoverall predictive success rate was 70%, a result that was slightlylower than the previously published studies, which cite the predictiverate as ˜76% (Groot, et al. (2011) Immunome Res. 7:2-7; Moise, et al.(2013) Humm. Vaccin. Immunother. 9:2060-2068). Allele-specificexamination revealed that the predictions were in the previouslyobserved range for DRB1*0101, 0301, 0401, and 0701. No data could befound for 0801. It was observed that for 1301 the majority of peptidesregistered as either weak or non-binders. This observation would suggestthat the test peptide used for 1301 was likely a really strong binder,and as such had skewed the data toward a smaller predictive successrate. Similarly, the predictive rate for 1101 and 1501 was lower thanpreviously reported and was also thought to have contributed to thelower overall rate.

The cluster 1 wild-type (C1WT) epitope was predicted to bind four out ofeight MHC II alleles tested. In accordance with the prediction, bothAsn12Gly and Asn13His mutations disrupted MHC II binding. Asn13Hismutation was better at removing DRB1*0801 and 1101 epitopes, and themutations were equally good at removing DRB1*1501 epitope. However, themutations also resulted in strong binding for DRB1*0101 (Asn12Gly), andweak binding for DRB1*1301 (Asn13His) (Table 12).

TABLE 12 IC₅₀, μM Pep- (No. of predicted eptitopes) tide 0101 0301 04010701 0801 1101 1301 1501 C1WT¹  214.9 >250 >250 >250   13.73 0.94 136.70  101.50 (0*) (0*) (0*) (0*) (2) (1*) (1) (1) N12G²<0.01 >250 >250 >250   58.63 5.16 >250 >250 (0) (0*) (0*) (0*) (1) (1*)(0*) (0*) N13H³ >250 >250 >250 >250 >250 4.13   41.63 >250 (0*) (0*)(0*) (0*) (1) (0) (1) (1) ¹SAQWLNNYKKGYGYG (SEQ ID NO: 148).²SAQWLGNYKKGYGYG (SEQ ID NO: 221). ³SAQWLNHYKKGYGYG (SEQ ID NO: 222).*Positive correlation between predicted binders and experimentallyobserved binders. Binding thresholds set to 5% for predictions and 10 μMfor experiments.

In the case of cluster 2 wild-type (C2WT) epitope, binding was predictedfor seven MHC II alleles, with multiple epitopes for DRB1*0101, 0301,0401, 0701, and 1501 (Table 13). The mutations were predicted to targetthe seven alleles except for DRB1*0101 and 0701, and for those threealleles (in addition to 0401) an increase in binding was observed. Oneexception was the Lys46His mutant, which showed a two-fold decrease inbinding to 0101. Lys46His also reduced binding to 1101, while Ile41Gluand Ile41Glu/Lys46His resulted in strong binding. All mutants had loweraffinity for 0301, as predicted. Ile41Glu and Lys46His showed reducedbinding to 1501, while Ile41Glu/Lys46His emerged as a strong binder. Thecombination of mutations was particularly bad as it resulted inincreased binding for all alleles but 0301. The high binding affinity ofthis cluster could be explained by the large number of epitopes. Out ofa total of 13 epitopes across the eight alleles, the mutations werepredicted to disrupt only four epitopes. Furthermore, this particularregion contained an EpiBar, and previous studies show that theseepitopes tend to be more immunogenic than epitopes that do not haveEpiBars (Groot, et al. (2011) supra).

TABLE 13 IC₅₀, μM (No. of predicted eptitopes) Peptide 0101 0301 04010701 0801 1101 1301 1501 C2WT¹  0.22   60.10 13.73   8.19 >250  10.08 103.90   2.37 (2*) (2) (3) (2*) (0*) (1) (1) (2*) 141E²  0.59 >250 3.48  7.51 >250 146.10  142.60  7.44 (2*) (1) (3*) (2*) (0*) (0*) (0*)(1*) K46H³  0.49  151.62  1.65  4.46  87.48  18.29  212.00  2.55 (2*)(1) (2*) (2*) (0*) (1) (0*) (2*) 41_46⁴ <0.01 >250 <0.01 14.33 >250  <0.01 >250 <0.01 (2*) (1) (2*) (2) (0*) (1*) (0*) (1*)¹GVDFFMNIGTPVKAISSGKIV (SEQ ID NO: 223). ²GVDFFMNEGTPVKAISSGKIV (SEQ IDNO: 224). ³GVDFFMNIGTPVHAISSGKIV (SEQ ID NO: 225).⁴GVDFFMNEGTPVHAISSGKIV (SEQ ID NO: 226). *Positive correlation betweenpredicted binders and experimentally observed binders. Bindingthresholds set to 5% for predictions and 10 μM for experiments.

The cluster 3 wild-type (C3WT) epitope was a predicted binder ofDRB1*0101, 0701, and 1101, with multiple epitopes for 0801 and 1501(Table 14). C3WT was experimentally shown to also weakly bind 1301.Leu83Met mutation was anticipated to disrupt binding in only 0801 and1501. In agreement with the prediction, an approximately five-folddecrease in binding for 0801 was found. However, more than a two-folddecrease in 0701 (strong to moderate), ten-fold reduction for 1101, anda ˜2000-fold drop for 0101 (strong to weak) was also observed. Increasedbinding was detected for 1501 (opposite of what was predicted), 1301,and 0301. For 1501, a shift from a weak to a moderate binder wasobserved, while 1301 remained a weak binder, and 0301 a non-binder.Thus, the Leu83Met mutation was largely productive as it reduced bindingto four alleles and did not cause a change of classification for twoother alleles (peptides remained weak or non-binders).

TABLE 14 IC₅₀, μM (No. of predicted eptitopes) Peptide 0101 0301 04010701 0801 1101 1301 1501 C3WT¹ <0.01 155.43  38.12 0.41 0.14 <0.01 54.699.51 (1*) (0*) (0*) (1*) (2*) (1*) (0*) (2*) L83M² 32.39 114.83 >2502.09 0.37  0.11 27.38 4.42 (1) (0*) (01) (1*) (1*) (1*) (0*) (1*)¹GVHRQWYMIILSKYNVKVGD (SEQ ID NO: 149). ²GVHRQWYMHMSKYNVKVGD (SEQ ID NO:150). *Positive correlation between predicted binders and experimentallyobserved binders. Binding thresholds set to 5% for predictions and 10 μMfor experiments.

The cluster 4 wild-type (C4WT) epitope was predicted to bind three outof eight MHC II alleles tested: DRB1*0101, 0401, and 0801. In additionto 0101 and 0401, binding was observed for 0301 (weak), 1101 (strong),1301 (weak), and 1501 (strong), but not for 0801 (Table 15). Inaccordance with the prediction, the mutations disrupted MHC II binding.One exception was mutation Lys95Glu, which was predicted to reducebinding to 0101 and 0801, but instead resulted in stronger binding (orremained as a strong binder) across all eight alleles. The mutationssignificantly decreased binding to 0101: Tyr93His, Ile99Gln, andTyr93His/Lys95Glu/Ile99Gln transformed a strong binder into a weak ornon-binder, and Tyr93His/Lys95Glu turned a strong binder into a moderateone. All mutations and their combinations reduced the binding for 0301from weak to non-binders. Similarly, all mutations but Tyr93His/Ile99Glnresulted in significant decreases in binding to 1101 (strong toweak/non-binder), and all mutants except Tyr93His/Lys95Glu lessened thebinding for 1301. Lastly, only Ile99Gln and Tyr93His/Lys95Glu/Ile99Glneliminated binding to 1501 (strong to weak/non-binder). MutationsIle99Gln, Tyr93His/Ile99Gln, and Tyr93His/Lys95Glu slightly increasedbinding affinity for 0701 by turning a non-binder C4WT into a weakbinder. Overall, mutations Tyr93His, Ile99Gln, Tyr93His/Lys95Glu,Lys95Glu/Ile99Gln, and Tyr93His/Lys95Glu/Ile99Gln were the mostproductive across all eight alleles. Tyr93His/Lys95Glu/Ile99Gln wasparticularly good, as it eliminated binding to all eight alleles.

TABLE 15 IC₅₀, μM (No. of predicted eptitopes) Peptide 0101 0301 04010701 0801 1101 1301 1501 C4WT¹  <0.01  31.40   <0.01  92.86   62.54   1.69   78.47   <0.01 (1*) (0*) (1*) (0*) (1) (0) (0*) (0) Y93H²102.80 >250 >250 181.90 >250 >250 >250   <0.01 (0*) (0*) (1) (0*) (0*)(0*) (0*) (0) K95E³  <0.01   1.90   <0.01   4.81   12.24    6.32   25.69  <0.01 (0) (0) (1*) (0) (0*) (0) (0*) (0) I99Q⁴  31.74 >250    6.16 93.12 >250   24.32 >250   66.06 (0*) (0*) (0) (0*) (1) (0*) (0*) (0*)93_99⁵  <0.01 >250   <0.01  60.49 >250 >250 >250   <0.01 (0) (0*) (0)(0*) (0*) (0*) (0*) (0) 93_95⁶   9.98 110.20   25.52  24.94   30.93  25.02   18.92    0.06 (0) (0*) (1) (0*) (0*) (0*) (0*) (0) 95-99⁷ <0.01 >250   25.77 103.70 >250   25.43 >250   <0.01 (0) (0*) (0*) (0*)(0*) (0*) (0*) (0) 939599⁸ >250 >250 >250 179.30 >250 >250 >250 >250(0*) (0*) (0*) (0*) (0*) (0*) (0*) (0*) ¹DYVKAGQIIGWSGSTGY (SEQ ID NO:151). ²DHVKAGQIIGWSGSTGY (SEQ ID NO: 152). ³DYVEAGQIIGWSGSTGY (SEQ IDNO: 153). ⁴DYVKAGQQIGWSGSTGY (SEQ ID NO: 154). ⁵DHVKAGQQIGWSGSTGY (SEQID NO: 155). ⁶DHVEAGQIIGWSGSTGY (SEQ ID NO: 156). ⁷DYVEAGQQIGWSGSTGY(SEQ ID NO: 157). ⁸DHVEAGQQIGWSGSTGY (SEQ ID NO: 158). *Positivecorrelation between predicted binders and experimentally observedbinders. Binding thresholds set to 5% for predictions and 10 μM forexperiments.

The cluster five wild-type (C5WT) epitope was predicted to bind alleight alleles, with multiple epitopes for all except DRB1*0301 and 0801.Binding was experimentally observed in all but 0301 and 1301, with 0701and 0801 measuring as weak binders (Table 16). The most deimmunizingmutations were Arg118Thr/Ser122Asp/Ser124Gly andAsn121Gly/Ser122Asp/Ser124Gly, as they each significantly reducedbinding affinity to six alleles. For the 0301 allele, the IC₅₀ values ofthe mutants decreased slightly, but the peptides remained asnon-binders. This result agreed with the EpiSweep prediction, in whichthe mutations disrupted binding for all eight alleles. Asn121Glydiminished binding to four alleles, while Ser124Gly andAsn121Gly/Ser122Asp reduced binding to three. Asn121Gly/Ser122Asp alsoslightly increased the binding to 0301 (non-binder to weak binder).Similarly, Arg118Thr and R118T/S122D lessened the affinity to twoalleles, but showed an increase in binding to 0301 (non-binder to weakbinder). The observed increase in binding affinity for 0301 allele inmutation combinations could be explained by the fact that Ser122Asp waspredicted to introduce one epitope for the allele. In fact, it wasobserved that Ser122Asp changed a non-binder peptide C5WT into amoderate binder.

TABLE 16 IC₅₀, μM (No. of predicted eptitopes) Peptide 0101 0301 04010701 0801 1101 1301 1501 C5WT¹  0.15 >250  0.05   15.23   22.01<0.01 >250 <0.01 (2*) (1) (3*) (2 (1) (3*) (3) (3*) R118T² <0.01   68.39<0.01   23.14   57.70 <0.01 >250 <0.01 (2*) (1) (3*) (2) (1) (3*) (3)(3*) N121G³ <0.01 >250  6.13   17.40 >250  6.41 >250 <0.01 (2*) (1) (2*)(2) (1) (2*) (2) (3*) S122D⁴ <0.01    9.42 <0.01 >250  150.00 <0.01 >250<0.01 (1*) (2*) (2*) (0*) (1) (1*) (0*) (1*) S124G⁵ <0.01 >250  0.04 150.70 >250  0.30 >250 <0.01 (2*) (1) (2*) (2) (1) (2*) (3) (3*) 18_22⁶<0.01   28.16 <0.01  110.60 >250 <0.01 >250 <0.01 (1*) (1) (2*) (0*)(0*) (1*) (0*) (0) 182224⁷  7.43  132.90  7.52 >250 >250 14.13 >250 0.42 (1*) (0*) (2*) (0*) (0*) (1) (0*) (0) 21_22⁸ <0.01   77.42<0.01 >250  210.10 28.54 >250 <0.01 (1*) (1) (2*) (0*) (0*) (1) (0*) (0)212224⁹  0.18  116.40  5.55 >250 >250 13.45 >250 24.20 (1*) (0*) (2*)(0*) (0*) (1) (0*) (0*) ¹HLHFQRMVNSFSNPTAQ (SEQ ID NO: 159).²HLHFQTMVNSFSNPTAQ (SEQ ID NO: 160). ³HLHFQRMVGSFSNPTAQ (SEQ ID NO:161). ⁴HLHFQRMVNDFSNPTAQ (SEQ ID NO: 162). ⁵HLHFQRMVNSFGNPTAQ (SEQ IDNO: 163). ⁶HLHFQTMVNDFSNPTAQ (SEQ ID NO: 164). ⁷HLHFQTMVNDFGNPTAQ (SEQID NO: 165). ⁸HLHFQRMVGDFSNPTAQ (SEQ ID NO: 166). ⁹HLHFQRMVGDFGNPTAQ(SEQ ID NO: 167). *Positive correlation between predicted binders andexperimentally observed binders. Binding thresholds set to 5% forpredictions and 10 μM for experiments.

To compare immunogenicity between the full-length designs, the aggregateepitope score for each design was calculated (FIG. 3). This analysisshowed that the wild-type lysostaphin catalytic domain had a total of 26binding interactions (or epitopes): 14 strong, 1 moderate, and 11 weak.The enzyme also had 14 non-binding interactions. In comparison, 18 ofthe designs had an epitope score lower than the wild-type, six had thesame score, and only four had epitope scores that exceeded the wild-type(by maximum of two epitopes). All but three of the designs also had ahigher number of non-binding interactions as compared to the wild-type.The minimum number of epitopes was 18, and it was present in three ofthe variants: Flex 4* (5 mutations), Flex 11* (7 mutations), and Flex13* (6 mutations). Furthermore, 18 of the designs showed a reduction inthe number of strong binders, as compared to the wild-type.

A strong negative correlation was noted between the number of mutationsand the number of experimentally observed strong binders (Pearsoncoefficient −0.69). Similarly, a positive correlation was observedbetween the epitope score and the number of strong binders (Pearsoncoefficient 0.52). At the same time, no correlation was found betweenthe mutational load/epitope score and the total number of binders. Thisresult indicated that the algorithm was not only reducing binding, butwas also primarily targeting the strong epitopes.

In general, more deimmunized plans were found among flexible than rigidbackbone designs. On average, there were fewer strong, moderate, weak,and total binding interactions found in flexible than rigid plans. Thistrend may be explained in part by the fact that most rigid designs werereverted and missing one mutation. The lowest number of strong binders,eight, was observed in Flex 11* and Rigid 5*. As expected, the mostaggressive plan, Flex 9, showed a significant decrease inimmunogenicity, with a total of only 22 binding interactions: 9 strong,6 moderate, and 7 weak.

In Vitro Analysis of Flex 5 and Flex 9 Variants

Variants Flex 5 and Flex 9 were expressed, purified, and characterizedin biological duplicate. As an additional control, wild-type LST wasobtained from a commercial supplier and analyzed in parallel. Theapparent melting temperatures of both variants were consistent withvalues obtained during preliminary testing, but their specific rates ofbacterial lysis were found to be somewhat higher upon more rigorousanalysis (Table 17). Importantly, the deimmunized variants wereequivalent to or better than commercially sourced LST in both assays.The enzymes' antibacterial activity was further quantified by assessingminimal inhibitory concentration (MIC) toward four strains of S. aureus.The Flex 5 MIC for strain SA113 was equivalent to that of wild-type andcommercial LST, and it was within a single 2-fold serial dilution forthree clinical isolates, including MRSA strain 3425-3. Variant Flex 9also retained good bactericidal/bacteriostatic activity, preventingoutgrowth of all four strains at 200 ng/ml (˜7 nM) or less. Given thefact that the LST^(CAT) variants encoded four or eight mutations,respectively, their high levels of anti-staphylococcal activity werestriking.

TABLE 17 % WT MIC (μg/mL) Design Lytic Tm Strain Strain Strain Strain IDRate (° C.) SA113 6445 3425-1 3425-3 Wild- 100 ± 30  59.0 ± 0.02 ± 0.03± 0.04 ± 0.03 ± type 0.4 0.00 0.01 0.02 0.02 Commer- 60 ± 20 47.3 ± 0.03± 0.03 ± 0.04 ± 0.03 ± cial 0.4 0.00 0.00 0.01 0.00 Flex 5 70 ± 30 55.8± 0.02 ± 0.06 ± 0.11 ± 0.05 ± 0.2 0.01 0.04 0.09 0.03 Flex 9 60 ± 2052.8 ± 0.04 ± 0.13 ±  0.2 ±  0.2 ± 0.3 0.01 0.07 0.1  0.1  *Errors arestandard deviation from a minimum of biological duplicates measured intriplicate

In Vivo Efficacy and Immunogenicity of Flex 5 and Flex 9 Variants

To assess antibacterial activities in a more clinically relevantfashion, a murine lung infection model was employed, which uses an S.aureus clinical isolate. Mice were infected with live bacteria viaoropharyngeal aspiration, and one hour later they were treated via thesame route with a solution containing 2.5 μg of wild-type LST, variantFlex 5, or variant Flex 9. Twenty-four hours post-infection, mice weresacrificed, lungs were harvested, and viable bacterial counts weredetermined by plating serial dilutions of lung homogenate. All threeenzymes yielded a statistically significant 10-fold reduction inbacterial burden relative to a saline buffer control (one-way ANOVAP=0.007, Tukey post test), but there was no significant differencebetween the three treatments (FIG. 4A). Thus, the deimmunized candidatesretained wild-type efficacy in the infected and inflamed lungenvironment.

In vivo immunogenicity was evaluated using NOD/SCID/γ_(c) ^(−/−) micethat had been surgically humanized with human immune cells, livertissue, and thymus tissue (HUMI mice). Following transplantation ofhuman tissues at six weeks of age, HUMI mice were allowed to mature anddevelop circulating repertoires of human B and T cells. At 14 weekspost-transplantation, mice were divided into three groups of four eachand immunized subcutaneously with 100 μg of wild-type LST, Flex 5, orFlex 9 in adjuvant. Thirteen days post-immunization, mice weresacrificed, splenocytes were harvested and pooled for each group, andthe pooled cells were subjected to ex vivo restimulation with theircognate proteins. Cell proliferation was measured by tritiated thymidineuptake at 72 hours. The stimulation index (protein vs. DMSOproliferative response) was less than 2-fold for wild-type LST (FIG.4B), but it bears noting that T cells from humanized mice are widelyknown to exhibit impaired function. In particular, humanized mousesplenocytes have been shown to exhibit poor ex vivo proliferativeresponse even in the presence of potent stimulatory agents such asphytohaemagglutinin, ionomycin-PMA, and anti-CD3/anti-CD28 antibodycocktails (Watanabe, et al. (2009) Internatl. Immunol. 21:843-858).Moreover, following two to three in vivo immunizations with the powerfulantigen keyhole limpet hemocyanin in complete Freund's adjuvant (CFA),restimulated humanized mouse splenocytes fail to produce IFN-γ or IL-4(Watanabe, et al. (2009) Internatl. Immunol. 21:843-858) and exhibitonly a 2- to 6-fold stimulation index ex vivo (Tonomura, et al. (2008)Blood 111:4293-6). Thus, the significant (P=0.0005, two way ANOVA)1.6-fold stimulation index of the wild-type LST splenocytes is areasonable indicator of an antigen specific immune response,particularly given the fact that the mice of the current study receivedbut one immunization. Relative to the wild-type immunized group, pooledsplenocytes from Flex 5 and Flex 9 immunized mice exhibitedsignificantly reduced proliferation (FIG. 4B). After backgroundsubtraction, Flex 5 pooled cells showed a 50% reduced response and Flex9 pooled cells a 65% reduced response.

In addition to inherent immunogenicity in the context of a naïve immunesystem, the extent to which a deimmunized protein might evade anestablished memory response directed against the native sequence wasalso considered. Due to the long time-frame of such a study and theshort lifespan of HUMI mice, the memory response in transgenic DR4 micewas assessed. This homozygous strain has an intact murine immune system,with the exception that they bear a chimeric class II MHC based on thepeptide binding domains from human HLA DRA and DRB1*0401 (Ito, et al.(1996) J. Exp. Med. 183:2635-44). This stable transgenic model has anormal, healthy lifespan enabling extended studies, yet its antigenpresenting cells exhibit human peptide binding specificity. Ten DR4 micewere immunized and repeatedly boosted with sub-cutaneous injections ofwild-type LST. Nineteen weeks after the final boost, they were dividedinto two groups of five each such that each group exhibited similaraverage antibody titers. Mice were then rechallenged with either 100 μgwild-type LST or 100 μg variant Flex 5. Thirteen days later, splenocyteswere harvested, pooled for each group, and subjected to ex vivorestimulation with the cognate protein from the final rechallenge.Similar to the results in the HUMI mice, ex vivo restimulation of DR4splenocytes with wild-type LST yielded a 1.8-fold stimulation index(FIG. 4C). It bears noting that similarly small stimulation indices inDR2, DR3, and DQ8 transgenic mice have been shown to correlate withantigen specific antibody production and to be indicative of antigenspecific immune responses (Depil, et al. (2006) Vaccine 24:2225-9).Thus, the significant (P=0.0002, two way ANOVA) 1.8-fold stimulationindex seen here was a reasonable indicator of an anti-drug immuneresponse. In contrast to wild-type LST rechallenged mice, proliferationof pooled splenocytes from the Flex 5 challenge group was at or belowbackground levels (FIG. 4C). Thus, immune cells primed to recognizewild-type LST exhibited reduced activity upon rechallenge with Flex 5,indicating that the deimmunized variant effectively evaded the memoryresponse directed against the wild-type enzyme.

Example 2: Deimmunization of Lysostaphin Catalytic and Cell Wall BindingDomains Against HLA Allele DRB1*0401 Overview

This analysis was carried out to demonstrate that depletion of putativeT cell epitopes in LST would mitigate the anti-drug antibody responseand consequently enhance therapeutic efficacy. Epitope depleted variantswere developed using two distinct computationally-guided strategies:structure-based design of individual deimmunized variants followed byempirical improvement (designated “opt” variants) and structure-baseddesign and screening of combinatorial libraries enriched in functionallydeimmunized members (designated “lib” variants). HumanizedHLA-transgenic mice were used to assess the efficiency with which eachmethod deleted putative immunogenic epitopes and thereby preventedformation of anti-LST antibodies in vivo. Subsequently, a recurrentbacteremia model was used to gauge the extent to which LSTdeimmunization enabled clearance of systemic S. aureus infections. Thissystematic comparison between deimmunized variants and their wild-typecounterpart provides direct experimental evidence of the clinicallyrelevant connections between putative T cell epitopes, in vivoimmunogenicity, and therapeutic efficacy.

As a proof of concept, focus was placed on allele DRB1*0401 (hereafterDR4), which is highly prevalent in North American and Europeanpopulations. At a 5% threshold (i.e., peptides among the top 5% ofpredicted binders), the ProPred analysis tool (Singh & Raghava (2001)Bioinformatics 17:1236-7) predicted 16 DR4 restricted T cell epitopeswithin wild-type LST (LST^(WT) epitope score=16). The peptide epitopeswere arrayed as both overlapping clusters and isolated nonamersdistributed throughout the protein's sequence and structure (FIG. 5).Interestingly, ProPred predicted more epitopes for DR4 than for any ofthe seven other representative DRB1 alleles: 0101, 0301, 0701, 0801,1101, 1301, and 1501. Considering any single allele, therefore, the DR4model represented a high bar for global protein redesign.

Materials and Methods

Materials.

Primers were ordered with standard desalting from IDT Technologies(Coralville, Iowa). Restriction enzymes and Phusion DNA polymerase formolecular cloning were purchased from New England Biolabs (Ipswich,Mass.). All other reagents and supplies were from VWR Scientific(Philadelphia, Pa.), unless specifically noted.

P. pastoris expression vector pPIC9 and P. pastoris strain GS115(his4)were purchased from Invitrogen (Grand Island, N.Y.). E. coli DH5□ [F−Φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rK−, mK+) phoAsupE44 λ− thi-1 gyrA96 relA1], S. aureus strain SA113, and MRSA strainUSA400 were from the American Type Culture Collection (Manassas, Va.).

LST Homology Models.

As the crystal structure of lysostaphin was not available, homologymodels were constructed for the two domains. Three template structuresfrom LytM were selected for the catalytic domain (2B0P:A, 2B44:A and1QWY:A) (Firczuk, et al. (2005) J. Mol. Biol. 354:578-90; Odintsov, etal. (2004) J. Mol. Biol. 335:775-85). The three template structuresshare significant sequence identity (95%-97%), yet they have highlymobile loops (Firczuk, et al. (2005) J. Mol. Biol. 354:578-90) aroundconserved active sites. The highest sequence identity against thelysostaphin catalytic domain was 46.7%, which is sufficient to buildquality model structures (Baker & Andrej (2001) Science 294:93-96).Initial homology models were built using MODELLER. A template-lessregion (²⁴ PLGINGG ³⁰; SEQ ID NO:168) was modeled using a loop modelingmethod, FREAD (Choi & Deane (2010) Proteins 78:1431-1440), whichselected a sequence-similar loop from 1GMN:A (¹⁸⁰ PRGEEGG ¹⁸⁶; SEQ IDNO:169) (Lietha, et al. (2001) EMBO J. 20:5543-5555). The cell wallbinding domain was constructed using a single template structure(1R77:A, cell wall targeting domain structure of ALE-1, a lysostaphinhomolog) with a high sequence identity (83.5%) (Lu, et al. (2006) J.Biol. Chem. 281:549-558). The best models were selected in terms of theDOPE statistical potential function score (Shen & Sali (2006) ProteinSci. 15:2507-2524). The catalytic domain models were minimized againstAMBER99sb with GB/SA in order to relax the predicted loop (Hornak, etal. (2006) Proteins 65:712-725; Still, et al. (1990) J. Am. Chem. Soc.112:6127-6129).

Preprocessing of Mutation Choices.

For each domain, three iterations of PSI-BLAST (Altschul, et al. (1997)Nucleic Acids Res. 25:3389-3402) were run against the non-redundantdatabase to find homologs. Multiple sequence alignments were constructedfor the identified sequences, and processed to identify those that werenot too gappy (at most 25%), and were sufficiently similar to LST (atleast 35%) but sufficiently different from each other (at most 90%identical). A set of 114 representative catalytic domain homologs and 23representative cell wall binding domain sequences remained. Amino acidswere identified at each position in each multiple sequence alignment andsubsequently used as possible mutations for design. Particular positionsand mutations deemed functionally important were not allowed to mutate(positions 32, 36, 82, 113, 115, 117, 118, 119, 125, 126, 127 werelocked down; specific mutations Y33T, F38A, F38G, M39A, I41R, S124Y andR118T were disallowed). A second filtering step kept only thosemutations predicted to delete at least one epitope, as determined usingProPred at a 5% threshold. To avoid presumed deleterious effects,mutations to and from proline and cysteine were also excluded.

The mutational choices were expanded for library design, since screeningallows for riskier substitutions. In particular, Chou-Fasman (Chou &Fasman (1974) Biochem. 13:222-45) propensities were used to identifyresidues likely to be acceptable in the wild-type secondary structureenvironment, according to a stringent threshold of 1.5. In addition,since the library was constructed with degenerate oligonucleotides thatcan incorporate additional amino acids beyond the desired ones, theseadditions were allowed, as long as the desired to undesired ratio withina degenerate oligonucleotide remained above 2:3.

Structure-Based Design of Individual Variants. In order to generateepitope depleted designs, the structure-based deimmunization methodEpiSweep was applied to each domain as described in Example 1. OSPREY(ver. 2.0) (Chen, et al. (2009) Proc. Natl. Acad. Sci. USA106:7678-7678) was used to assess one- and two-body energy terms forpossible rotamers (Lovell, et al. (2000) Proteins 40:389-408) for themutational choices, according to the AMBER force field (Pearlman, et al.(1995) Comput. Phys. Commun. 91:1-41) and a reference energy (Lippow &Tidor (2007) Curr. Opin. Biotechnol. 18:305-311). ProPred (Singh &Raghava (2001) Bioinformatics 17:1236-7) at a 5% threshold was used toassess DR4 epitope (HLA Allele DRB1*0401) content, characterizing each9mer as either a binder or a non-binder. It was found that at leastseven mutations were required to deplete all the predicted DR4 epitopesin the catalytic domain; six were required to fully deplete the cellwall binding domain. A set of 20 energy optimal and near-optimal fullydepleted designs were identified for each domain via the EpiSweepoptimization algorithm. The most energy optimal catalytic domain designcontained a mutation at Ser124, which analysis has identified as beingpotentially detrimental. However, the alternative double mutationAsn121Gly and Ser122Gly is predicted to remove the same epitope, yetthese two mutations manifest higher experimentally determined activitythan LST^(WT). The energy difference between the most energy optimaldesign (containing a detrimental Ser124 mutation) and the alternativedouble mutation design was marginal (−294.94 and −293.9). Thus, a design(Opt1) was selected so as to possess the most energy optimal design forthe cell wall binding domain and the alternative double mutation in theC-terminal portion of the catalytic domain (Asn121Gly and Ser122Gly).

Structure-Based Design of Deimmunized Libraries.

In order to design combinatorial libraries enriched in stable,deimmunized variants, a method called EpiSOCoM was developed, whichaugments the SOCoM structure-based library design approach with epitopeanalysis, and employs a sweep-based Pareto optimization algorithm(Parker, et al. (2013) J. Comput. Biol. 20:152-65) to simultaneouslyoptimize both energy and epitope content. Given a set of possiblepositions at which to mutate and possible amino acids to incorporate atthose positions, along with a desired library size, EpiSOCoM selects asubset of the positions and subsets of the substitutions at thosepositions. It thereby specifies the construction of a library composedof all combinations of the substitutions and corresponding wild-typeresidues. EpiSOCoM optimizes a library for the average energy score andthe average epitope score over its constituent variants, so that ingeneral variants will be “good”. Its Pareto optimization algorithmidentifies all library designs (positions and substitutions) makingundominated trade-offs between the two scores, in that no other librarydesign is better for both.

In order to enable rapid assessment of structure-based energies withoutexplicit rotameric modeling of each variant, SOCoM employs a ClusterExpansion (Grigoryan, et al. (2009) Nature 458:859-64; Grigoryan, et al.(2006) PLoS Comput. Biol. 2:e63) (CE) technique that expressesstructure-based properties in terms of a protein-specific function ofamino acid sequence. Here, Rosetta (Rohl, et al. (2004) Methods Enzymol.383:66-93) was employed in a preprocessing step to model and evaluateenergies of random LST variants based on the allowed mutational choices.These structural training sets allowed CE to express the energy Ψ for apossible LST variant S via a sum over position-specific one- andtwo-body sequence potentials:

Ψ(S)=Σ_(i)ψ_(i)(a _(i))+Σ_(i,j)ψ_(i,j)(a _(i) ,a _(j))  (1)

where the sums involve amino acid a_(i) at position i and a_(j) atposition j. A total of 9000 catalytic domain and 6000 cell wall bindingdomain models were used to train CE models, following the CE guidelines.These were then able to make accurate predictions for the energies ofnew random variants in a non-overlapping testing set (of size ˜20% thatof the training set), achieving correlations between the CE sequencepotential and Rosetta energies of 0.8 for the catalytic domain and 0.9for the cell wall binding domain. This modeling step thus enables fast,accurate prediction of energies within the “inner loop” of designoptimization.

In order to assess a library without enumerating all its variants (nottractable when optimizing over the massive design space of possiblelibraries), SOCoM employs library-averaged position-specific scores. Forexample, if the set {Arg, Lys} were to be incorporated at one position,then the energetic contribution of that position, averaged over allvariants the library, would be the average of the Arg energy and the Lysenergy; similarly the pair-wise contribution from that position andanother incorporating {Asp, Glu}, averaged over the library, would bethe average of Arg:Asp, Arg:Glu, Lys:Asp, and Lys:Glu. Thus, given theallowed mutations, SOCoM precomputes ψ _(i), the average energeticcontributions of possible subsets of amino acids that could be chosen ata position i, and ψ _(i,j), the average for pair of positions i and j.It then evaluates the average energy Ψ over a whole library T with anequation analogous to that for a single variant:

Ψ(T)=Σ_(i) ψ _(i)(T _(i))+Σ_(i,j) ψ _(i,j)(T _(i) ,T _(j))  (2)

where the sums now involve sets of amino acids T_(i) at position i andT_(j) at position j. Thus assessment of a library within theoptimization is as efficient as assessment of a single variant.

To develop EpiSOCoM, it was also necessary to “lift” the epitope scoreto library-averaged contributions, in a manner analogous to SOCoM'streatment of energy scores. If amino acids (T_(i), T_(i+1), . . . ,T_(i+8)) were to be incorporated at the nine contiguous positionsstarting at i, then the average epitope score contribution ē from thevarious 9mer combinations of amino acids is calculated as:

$\begin{matrix}{{\overset{\_}{e}}_{i} = \frac{{\sum_{{a_{i}e\; \tau_{i}},{a_{i + 1}e\; \tau_{i + 1}\ldots}\mspace{11mu},{a_{i + 1}e\; \tau_{i + 1}}}{e\left( {a_{i}a_{i + 1}\mspace{14mu} \ldots \mspace{14mu} a_{i + s}} \right)}}\;}{{T_{i}}{T_{i + 1}}\mspace{14mu} \ldots \mspace{14mu} {T_{i + 2}}}} & (3)\end{matrix}$

where the sum is over each combination of amino acids, one from eachset, and the function e(⋅) gives the epitope score of the 9mer. Then theaverage epitope score, Ξ, of the library is simply the sum over all9mers:

Ξ=Σ_(i=1) ^(n−8) ē _(i)  (4)

SOCoM uses an integer linear programming formulation to choose anoptimal set of positions and sets of amino acids so as to optimize Eq. 2subject to library size constraints. With EpiSOCoM, there are twoobjectives, energy (Eq. 2) and epitope score (Eq. 4). Since there is noa priori means to determine the best balance between theseincommensurate properties, EpiSOCoM generates all Pareto optimal designsrepresenting the best balance, enabling subsequent characterization ofthe trade offs and selection of suitable designs. To identify Paretooptimal designs, it employs a sweep algorithm based on that of EpiSweep.At each step in the sweep, average library energy is optimized (Eq. 2)according to a constraint on the average epitope score (Eq. 4). Theconstraint is successively tightened, so that each library must have abetter epitope score (and thus worse energy) than the previous one. ThePareto optimization is implemented as an iterative layer over aconstrained version of SOCoM, which in turn uses the IBM CPLEX integerprogramming solver to optimize each design.

Protein Expression, Purification and Characterization.

LST and its derivatives were secreted from P. pastoris. Briefly,recombinant Pichia strains were cultured in a 2.5 L bioreactor (AppliconBiotechnology), and the proteins were captured from the supernatant bypolyethylene glycol-6000 (PEG-6000) precipitation and purified tohomogeneity by SP SEPHAROSE F.F. cation exchange chromatography.Endotoxin was removed from the protein preparation by TRITON-X114extraction (Liu, et al. (1997) Clin. Biochem. 30:455-63). Proteinexpression levels were estimated by densitometry analysis of SDS-PAGEgels. The activities of proteins were assessed by determination ofminimal inhibitory concentrations (MIC) against S. aureus strain SA113,and are reported as a normalized percentage relative to the MIC dilutiondetermined for LST^(WT) (i.e., 50% activity is 2-fold higher MICrelative to wild-type, and 25% activity is 4-fold higher MIC relative towild type).

Library Construction and Screening.

LST libraries were constructed by splice overlapping PCR with theprimers shown in Table 18.

TABLE 18 Sequence SEQ ID Mutations Primer (5′→3′) NO: Library A Y33NDY,Forward GGTATGCACDATGGTGTTGACTTCTYTATGAA 170 F38SF, CATCGGTAMGCCAGTCAAGT43KT Reverse CTTGACTGGCKTACCGATGTTCATARAGAAGT 171 CAACACCATHGTGCATACCI70KRI, Forward GGTTTGADAGAGAACGACGGTGWSCACAGACA 172 V75DEVV,AWSGTACATGCACTTGVGTAAGTAC W79TRSW, ReverseGTACTTACBCAAGTGCATGTACSWTTGTCTGT 173 S84SRG GSWCACCGTCGTTCTCTHTCAAACCV120DV, Forward CACTTCCAAAGAATGGWTAACDSKTTCTCCAA 174 S122TTRSA CTCCGCTAGGSSWC Reverse AGCGGAGTTGGAGAAMSHGTTAWCCATTCTTT 175 GGAAGTG Library BS166TTRS, Forward GGTACCTTGTACAAGASMGAGTCCGSCTCCTT 176 A169AG CACCCCAAACReverse GTTTGGGGTGAAGGAGSCGGACTCKSTCTTGT 177 ACAAGGTACC S191SG, ForwardTCCATGCCACAARGCGGTGDMTTGAAGGCTGG 178 V193EDGGVV, TCAAACCAYTCACTACGACGAGI200TI Reverse CTCGTCGTAGTGARTGGTTTGACCAGCCTTCA 179AKHCACCGCYTTGTGGCATGGA N219NDY, Forward GTDATTCCGGTSAGAGAATCTACTTGCCAGTC180 Q222QE, AGAACCTGGAACAAGTCCACCVAWAC N236KNQHED ReverseGTWTBGGTGGACTTGTTCCAGGTTCTGACTGG 181 CAAGTAGATTCTCTSACCGGAATHACLibrary C Y33NDY, Forward GGTGGTATGCACDATGGTGTTGACTTCTTTVK 182M39RMRLGV, GAACATCGGTAMGCCAGTCAAGGCT T43KT ReverseAGCCTTGACTGGCKTACCGATGTTCMBAAAGA 183 AGTCAACACCATHGTGCATACCACC W79TRSW,Forward GGTGAGCACAGACAAWSGTACATGCACTTGVG 184 S84SRG TAAGTACAACGTCAAGReverse CTTGACGTTGTACTTACBCAAGTGCATGTACS 185 WTTGTCTGTGCTCACC S1915G,Forward AGATCCATGCCACAARGCGGTGDCTTGAAGGC 186 V193DGV TGGTCAA ReverseTTGACCAGCCTTCAAGHCACCGCYTTGTGGCA 187 TGGATCT N236KNQHED ReverseATCGGAATTCTTACTTGATGGTACCCCACAAG 188 ACACCCAAGGTWTBGGTGGACTTGTTCLibrary D Y33NY, Forward GGTATGCACWATGRCGTTGACTTCTTTATGAA 189 G34DG,CATCGGTAMGCCAKTAAAGGCTATC T43KT, ReverseGATAGCCTTTAMTGGCKTACCGATGTTCATAA 190 V45VL AGAAGTCAACGYCATWGTGCATACCY33NDY, Forward GGTATGCACDATGGTGTTGACTTCTTTATGAM 191 N40KNIT,WATCGGTAVACCAGTCAAG T43KTR Reverse CTTGACTGGTBTACCGATWKTCATAAAGAAGT 192CAACACCATHGTGCATACC V35DEVV, Forward CACTATGGTGWSGACTTCTTTATGAACATCGG193 T43TKR, TAVACCAGDMAAGGCTATC V45EDGGVV ReverseGATAGCCTTKHCTGGTBTACCGATGTTCATAA 194 AGAAGTCSWCACCATAGTG F116SCF ForwardCCACACTTGCACTBCCAAAGAATGGAT 195 Reverse ATCCATTCTTTGGVAGTGCAAGTGTGG 196R186TR, Forward GGTCCATTCASGTCCATGCCACAAKCTGGTGT 197 S191AS CTTG ReverseCAAGACACCAGMTTGTGGCATGGACSTGAATG 198 GACC S191SG, ForwardTCCATGCCACAARGCGGTGDCTTGAAGGCTGG 199 V193DGV T ReverseACCAGCCTTCAAGHCACCGCYTTGTGGCATGG 200 A R186TTSRP ForwardGGTCCATTCNSSTCCATGCCACAAAGCGGTVD 201 RRAAGGSSCW, RTTGAAGGCT V193KKRRIMQQRRLLEE GGVV Reverse AGCCTTCAAYHBACCGCTTTGTGGCATGGASS 202 NGAATGGACCLibrary E N28NS, Forward GTATCARCGGTGGTATGCACTATGGCGTTGAC 203 I41NI,TTCTTTATGACCAWCGGTAMGCCAGT T43KT ReverseACTGGCKTACCGWTGGTCATAAAGAAGTCAAC 204 GCCATAGTGCATACCACCGYTGATAC G34DG,Forward CACTATGRCGTTGACTTCTTTATGACCAWMGG 205 I41KNII, TAMGCCAGTA T43KTReverse TACTGGCKTACCKWTGGTCATAAAGAAGTCAA 206 CGYCATAGTG G34EDGG, ForwardCACTATGRKGTTGACTTCTTTATGACCRDRGG 207 T43KTR, TAVACCAGTA I41KKRRIMReverse TACTGGTBTACCYHYGGTCATAAAGAAGTCAA 208 EEGGVV CMYCATAGTG N28NK,Forward GTATCAASGGTGRAATGCACTATGGCGTTGAC 209 G30EG,TTCTTTATGACCAWCGGTAVACCAGT I41NI, ReverseACTGGTBTACCGWTGGTCATAAAGAAGTCAAC 210 T43KRT GCCATAGTGCATTYCACCSTTGATACN121NK, Forward AGAATGGATAASACTTTCTSSAACTCCGCT 211 S124SSCW ReverseAGCGGAGTTSSAGAAAGTSTTATCCATTCT 212 T122TTIM, ForwardAGAATGGATAACAYRTTCTSSAACTCCGCTGC 213 S124SSCW T ReverseAGCAGCGGAGTTSSAGAAYRTGTTATCCATTC 214 T

The PCR products were ligated into pPIC9 vector and transformed intoDH5a. Constructs were sequence verified and transformed into P. pastorisby electroporation (Wu & Letchworth (2004) Biotechniques 36:152-4).Library construction and screening was implemented as an iterativedirected evolution strategy. Active library members were identifiedusing a moderate throughput plate halo formation assay. Briefly, P.pastoris transformants from each round of library construction werespread on YPM agar media (1% yeast extract, 2% peptone, 1% methanol, 1%agarose) and incubated at 30° C. for 2 days. Indicating top agarose(0.5% yeast extract, 1% peptone, 1% NaCl, 0.1 OD₆₀₀ SA113, 1% lowmelting agarose) was poured onto the YPM yeast plates, and the plateswere incubated at 37° C. for 10 hours. Yeast clones expressing activeenzymes were identified by their characteristic halo or zone ofclearance. Approximately 10,000 clones were screened for each round. Thegenes encoding the 10 variants exhibiting the largest halos were PCRamplified from the genomically integrated cassette, subcloned back intopPIC9, sequenced, and retransformed into freshly prepared P. pastoriscells for functional validation by determination of MIC. The mostdeimmunized and functional variant was used as the starting point forthe subsequent round of library construction and screening.

In Vivo Studies.

The protocols for animal infection, treatment, and immunization werecarried out to minimize animal suffering. C57Bl/6 mice were purchasedfrom the Jackson Laboratory (Bar Harbor, Me.). C57Bl/6 background AbbKnockout/Transgenic HLA-DR4 mice (B6.129S2-H2-Ab1^(tm1Gru)Tg(HLA-DRA/H2-Ea,HLA-DRB1*0401/H2-Eb) Kito) were purchased from TaconicFarms (Germantown, N.Y.).

In Vivo Immunogenicity.

A 100 μl volume of 100 μg purified wild-type or variant enzyme incomplete Freund's adjuvant (CFA) was injected subcutaneously in eitherDR4 (N=5 per group) or C57Bl/6 (N=4 per group) mice. Thirteen daysfollowing immunization, serum was collected and anti-LST IgG antibodytiters (specific to wild-type or variant protein) were measured byELISA. Briefly, wild-type or variant protein antigen was coated ontohigh binding ELISA plates, followed by blocking with BSA. Immune serumfrom mice was serially diluted into the coated plates, which were thenprobed using goat anti-mouse IgG-HRP conjugate (Santa CruzBiotechnology, Dallas, Tex.) at a working concentration of 1:1000.Plates were subsequently developed using TMB Substrate (Santa CruzBiotechnology) Reported titers were defined as the serum fold dilutionyielding an absorbance of 1.5.

In Vivo Efficacy.

Prior to bacterial challenge, the immune systems of DR4 mice (N=3 pergroup) were primed 3-times with weekly subcutaneous injections of 100 μgLST^(WT) or Lib5 variant in sterile phosphate buffered saline (PBS: 2.7mM KCl, 1.5 mM KH₂PO₄, 8.9 mM Na₂HPO₄, 136.9 mM NaCl, pH 7.4). Theseimmunizations and boosts contained no adjuvant. Anti-LST antibody titerswere determined as described above. For the first cycle of infection andtreatment, mice were challenged with intraperitoneal administration of2×10⁸ CFU Staphylococcus aureus strain USA400 in a 3% suspension ofporcine mucin, and one hour later were treated by intravenous tail veinadministration of 500 μg of LST^(WT) or Lib5 variant in sterile PBS.Mice that were rescued by the enzyme treatment underwent subsequentinfection and treatment cycles at weekly intervals. To compensate fordevelopment of innate murine antibacterial immunity following the firstbacterial exposure, mice were challenged with 1×10⁹ CFU USA400 in thesecond and third cycles, but the treatments remained at 500 μg of theappropriate protein. As a control to verify the lethal bacterial dose ineach cycle, one mouse was given a sham treatment of PBS.

Structure-Based Design of Individual Variants

The EpiSweep algorithm (Parker, et al. (2013) J. Comput. Biol.20:152-65) was used to optimize deimmunized variants, making the besttrade-offs between predicted reduction in epitope content, as evaluatedby ProPred, and predicted maintenance of protein stability, as evaluatedby structure-based rotamer energy. Panels of 20 fully depleted designs(i.e., DR4 epitope scores=0) were generated separately for both thecatalytic and cell wall binding domains. A variant combining low energydesigns from each domain, Opt1 (Table 19), was selected for experimentalanalysis and cloned into an optimized Pichia pastoris expression system.Unfortunately, this 14-mutation design failed to yield functionalprotein (Table 20).

TABLE 19 Residues in the catalytic domain Design Y33 F38 N40 I70 N72 V75S84 M119 V120 N121 S122 LST^(WT) Opt1 T S H Q Y R G G Opt2 T S H Q Y G GOpt3 T S H Q Y G G Opt4 T S H Q Y G G Lib1 K E D T Lib2 Lib3 K E D TLib4 K E G D T Lib5 T K E G D T Residues in the cell wall binding domainDesign S166 S168 A169 R186 S191 V193 I200 N219 S234 N236 LST^(WT) Opt1 EK W T Y D Opt2 E K W T Y D Opt3 T Y D Opt4 T Y K Lib1 Lib2 T G T Y Lib3T G T Y Lib4 T G T Y D Lib5 T G T A T Y D

TABLE 20 Mutation Epitope Expression Activity Design Load Score Level (%WT) (% WT) LST^(WT) 0 16 100  100 Opt1 14 0 ND^(b) ND^(b) Opt2 13 1ND^(b) ND^(b) Opt3 10 5 20 ND^(a) Opt4 10 5 10 0.25 Lib1 4 13 80 100Lib2 4 12 80 100 Lib3 8 8 60 50 Lib4 10 6 50 50 Lib5 13 3 50 50^(a)Activity measured as minimal inhibitory concentration (MIC),reported as the % fold dilution, relative to wild type, at which the MICwas achieved. ^(b)Not Detectable.

The LST redesign work in Example 1 shows that single mutations couldundermine otherwise stable and active deimmunized variants. Therefore,detrimental mutations in the Opt1 design were identified bysystematically reverting mutations and mutation combinations. Analysisof isolated Opt1 mutations revealed that Met119Arg single-handedlyabolished protein secretion, but reversion of this single mutation indesign Opt2 (Table 19) failed to restore expression (Table 20).Ultimately, it was found that the three mutation combination Ser166Glu,Ser168Lys, and Val193Trp also undermined expression, and reversion towild-type at these three sites as well as Met119 generated anexpressible 10-mutation variant, Opt3 (Table 20).

Although Opt3 achieved reasonable expression levels (Table 20), purityanalysis by SDS-PAGE revealed two bands: one at the expected 25 kDa massand a second at approximately 30 kDa. Based on the results in Example 1,it was suspected that a latent N-linked glycosylation sequon in theC-terminal cell wall binding domain (²³²NKS²³⁴) had been activated.Therefore, the deimmunizing Asn236Asp mutation was exchanged with theSer234Lys mutation, which deleted both the C-terminal epitope and theN-linked glycosylation sequon. The resulting 10-mutation variant, Opt4,bore only five predicted DR4 epitopes and expressed as a single 25 kDaband, albeit with lower yields than Opt3 (Table 20). Although the highlyengineered Opt4 variant was produced in a folded and secretion competentstate, it was subsequently found to possess only a small fraction of thewild type enzyme's antibacterial activity (Table 20).

Structure-Based Deimmunization Via Computational Library Design

In parallel to design of individual variants, an alternativecombinatorial approach was pursued in which computational design wasused to generate LST libraries predicted to be enriched in functional,deimmunized variants. The structure-based library design method SOCoMwas augmented with epitope analysis in order to identify residuepositions and mutations whose combinations yielded variants with lowepitope scores, as evaluated by ProPred, along with good energies, asevaluated by a Cluster Expansion potential trained on Rosetta models. Inthe initial round, the designs were based on the wild-type reference,while in succeeding rounds the reference was shifted to the lead cloneselected from the previous library screen.

Library A targeted nine sites in the catalytic domain, and thecomplementary Library B population targeted eight sites in the cell wallbinding domain (Table 18). Although the deimmunized LST design space wasmassive, the initial libraries were constrained to fewer than 40,000members so as to maintain some parity between library size and thescreening capacity of agar plate halo formation assays. Approximately10,000 clones were screened from both Libraries A and B, and 10large-halo-forming colonies from each were sequenced and functionallyvalidated. The most promising variant from Library A (clone Lib1)contained four mutations in the catalytic domain and exertedantibacterial activity equivalent to LST^(WT) (Tables 19 and 20).Likewise, the most promising variant from Library B (clone Lib2)possessed four mutations in the cell wall binding domain (Table 19) andalso had wild-type antibacterial activity (Table 20).

The respective deimmunized domains of variants Lib1 and Lib2 werecombined to produce variant Lib3, containing eight mutations (Table 19),a 50% reduction in predicted epitope content, and retention of 50%wild-type activity (Table 20). The structures of the two Lib3 domainswere subsequently modeled and used as templates in another round ofdeimmunized library design. The resulting Library C targeted five sitesin the catalytic domain and three sites in the cell wall binding domain(Table 19). Functional screening of 10,000 clones yielded variant Lib4,which deleted 10/16 DR4 epitopes. Relative to its Lib3 startingtemplate, Lib4 contained one additional mutation each in the catalyticand cell wall binding domains and retained equivalent antibacterialactivity (Table 20). The domains of Lib4 were used in another iterativeround of modeling and deimmunized library design, yielding Library D,from which 10,000 clones were screened to isolate variant Lib5. VariantLib5 deleted 13/16 putative DR4 epitopes (Table 19), yet retained 50%wild-type expression and 50% antibacterial activity (Table 20). Comparedto variant Opt4, the best enzyme from the individual protein designefforts, Lib5 exhibited both a lower predicted epitope score and highermeasured functionality.

Further library construction and screening efforts (Library E) failed toidentify additional functional constructs. Therefore, the 13-mutationLib5 variant was designated the lead candidate for further analysis. Itis interesting to note that the three putative epitopes remaining invariant Lib (³³YGVDFFMTI⁴¹, SEQ ID NO:215; ³⁸FMTIGTPVK⁴⁶, SEQ ID NO:216;and ¹¹⁶FQRMDNTFS²⁴, SEQ ID NO:217) either encompass or are adjacent toamino acids responsible for active site Zn²⁺ coordination (His32, Asp36and His115). This fact may explain the elusive nature of functionalmutations within these regions; screening of diverse library populationsidentified only one functional substitution in region 33-46 and two inregion 116-124 (Table 19).

Epitope Depleted Designs Display Significantly Reduced Immunogenicity InVivo

The extent to which epitope depletion impacted in vivo immunogenicitywas subsequently assessed. Anti-LST antibody responses were determinedin both C57Bl/6 and transgenic DR4 mice, the latter of which are nullfor endogenous murine MHC II, but bear a chimeric MHC II receptorderived from human HLA DRB1*0401 (Ito, et al. (1996) J. Exp. Med.183:2635-44). As a stringent benchmark for deimmunization, mice wereimmunized subcutaneously with protein in complete Freund's adjuvant, apowerful immunostimulant. Two weeks after a single immunization withLST^(WT), all DR4 mice mounted a potent anti-LST IgG antibody response,with titers between 1:150 and 1:1700. In contrast, mice immunized withOpt4 showed a striking reduction in titers; only 2/5 mice exhibited anydetectable anti-LST antibodies, and even those were substantiallyreduced relative to LST^(WT) immunized animals. Variant Lib5 alsoelicited a reduced antibody response; only one animal exhibited highantibody titers (1:1200), three showed significantly lower antibodytiters (1:15 to 1:26), and 1 mouse exhibited near background levels ofanti-LST antibodies. Importantly, both LST^(WT) and Opt4 were equallyimmunogenic in the C57Bl/6 laboratory mouse strain, while Lib5 wasactually more immunogenic than LST^(WT) (IgG titers 1:4400 to 1:15,000versus 1:560 to 1:2100, respectively). Thus, the striking reductions inOpt4 and Lib5 immunogenicity were specifically associated withdisruption of molecular recognition by human DR4, as opposed to thenative murine MHC II.

It was of note that although the protein design process was blinded toall but allele DR4, ProPred predictions indicated that neither Opt4 norLib5 contained neoepitopes for seven other representative human DRB1alleles (Supp. FIG. S1). In fact, over and above the 11 putative DR4epitopes deleted from Opt4, predictions suggested that 12 epitopesassociated with alleles DR1, DR3, DR7, DR13, and DR15 had also beendeleted. Similarly for Lib5, in addition to deletion of 13DR4-restricted epitopes, ProPred predicted deletion of 18 additionalepitopes associated with the other seven DRB1 alleles.

Lysostaphin Deimmunization Translates into Improved Therapeutic Efficacy

In some embodiments, therapeutic application of LST may require repeatedadministration to fully eradicate S. aureus infections. Therefore, tomore closely mimic potential clinical applications, the immunogenicityof LST^(WT) and Lib5 were monitored during weekly dosing in the absenceof adjuvant. Seven days after a third immunization, all DR4 micereceiving LST^(WT) had mounted a relatively strong immune response, withanti-LST titers in the 1:40 to 1:160 range. During the same time-frame,only one of three mice immunized with Lib5 developed high antibodytiters (1:120), where the other two Lib5 mice exhibited titers onlymarginally above background.

Using an S. aureus recurrent bacteremia model, the extent to which LSTimmunogenicity impacted in vivo efficacy was subsequently evaluated.Following determination of antibody titers at week three, the above DR4mice were infected by intraperitoneal administration of 2×10⁸ colonyforming units (CFU) of methicillin-resistant S. aureus (MRSA) strainUSA400. One hour later, mice were given a 500 μg intravenous bolus ofLST^(WT) or Lib5, respectively. Both enzymes rescued their respectivegroups from this initial infection, whereas a control mouse given a PBSsham treatment had to be sacrificed due to excessive morbidity.

One week later, antibody titers had increased for both the LST^(WT)group (1:300 to 1:650) and the Lib5 group (1 mouse >1:1000, with theremaining two between 1:15 and 1:20), but the latter continued toexhibit a lower overall trend. Mice were now infected with 10⁹ CFU ofMRSA and again treated 1 hour later with a 500 μg intravenous bolus ofthe respective enzyme. In this second infection cycle, where mice haddeveloped higher antibody titers, LST^(WT) failed to rescue any of thethree treated mice. Similarly, the single Lib5 mouse exhibiting highantibody titers succumbed to the infection, but the two lower titer Lib5mice were rescued from the second MRSA challenge.

The following week, antibody titers in the two surviving Lib5 mice werefound to have increased yet again (1:70 and 1:120), but notably theyremained below the week four LST^(WT) titers. Following a thirdinfection cycle with MRSA, one mouse was treated with Lib5 and survived,whereas the second mouse was given a PBS sham and succumbed to theinfection. As a whole, these results showed that humanized DR4 micemounted a strong immune response to LST^(WT), even in the absence ofadjuvant. During repeated administration, the weekly increase inanti-LST^(WT) antibody titers correlated with loss of efficacy.Conversely, immune responses were attenuated in two of three micereceiving the Lib5 deimmunized variant, and once again in vivo efficacytracked with anti-LST antibody titers. Lib5 exerted potent antibacterialefficacy against as many as three consecutive challenges with MRSA.

Throughout the study, anti-LST antibody titers below 1:256 correlatedwith enzyme-mediated rescue from S. aureus infection, whereas titersabove 256 were universally associated with failure of the antibacterialenzyme therapy. Variant Lib5's capacity to mitigate anti-drug antibodyresponses therefore manifested as enhanced efficacy relative toLST^(WT).

Example 3: Deimmunization of Lysostaphin Catalytic and Cell Wall BindingDomains Against HLA Alleles Representative of the HLA BindingSpecificity in Human Populations

To deimmunize LST against human HLA alleles DRB1*0101, 0301, 0401, 0701,0801, 1101, 1301, and 1501, individual variants and libraries predictedto be enriched in functional, deimmunized variants were generated.Epitope mapping of the catalytic domain (FIG. 6A) and cell wall bindingdomain (FIG. 6B) was carried out as described in Example 2. Using thisinformation, as well as solvent accessibility and evolutionaryconservation, deimmunized LST mutants were generated.

In particular, mutations in the cell wall binding domain (Table 21) werecombined with mutations in the catalytic domain of the Flex 9 mutant(Example 1) or a Flex 9 derivative.

TABLE 21 Residue Wild- Single Designs Library Designs Position type 1 21 2 160 Y N, H, D, Y 164 Y W 166 S E E K, N, T, R, S, E, D, A, G 168 S K169 A E, D, A, G 186 R T, R 193 V W D E, D, G, V D, G, V 195 K H H 200 IT T T, I T, R, I 209 D E, D, A, G 214 V I, M, V, L 215 G E, G 218 G D, GD, G 224 I K, R, I K, R, I 229 R R, G R, G 232 N Q Q 236 N D N, D 237 TK

Variants were screened and three variants of interest were identified,F11, F12 and F13 (Table 22).

TABLE 22 Variant Mutations^(#) SEQ ID NO: F11 N12G, I41E, L83M, K95E,I99Q, N121G, 218 S122D, S124G, S126P*, Y160H, A169G, R186T, N232Q* F12N12G, I41E, L83M, I99Q, N121G, S122D, 219 S124G, S126P*, Y160H, S166N,A169G, R186T, N232Q*, N236D F13 N12G, I41E, L83M, K95E, I99Q, N121G, 220S122D, S124G, S126P*, Y160H, S166N, A169G, R186T, N232Q*, N236D*aglycosylation mutations for efficient production in eukaryotic hostcells. These mutations are not required for expression in bacterialhosts. ^(#)Relative to wild-type lysostaphin sequence (SEQ ID NO: 49).

F11, P12 and F13 were screened for in vitro inhibitory activity againstMRSA strain USA400 (FIG. 7). This analysis indicated that variants F11and F13 retained 12.5% MIC activity relative to wild-type LST, whilevariant F12 retained 25% MIC activity relative to wild-type LST.Further, upon heating the variants at 50° C. for 1 hour prior todetermining activity against MRSA strain USA400, it was found thatwild-type LST retained full MIC activity, F13 retained 25% of itsoriginal MIC activity, and F12 retained 50% of its original MICactivity.

The in vivo efficacy of the F11 variant was further analyzed in C57BL/6mice. Mice were challenged with an intraperitoneal injection of 2×10⁸MRSA strain USA400, and 1 hour later the mice were treated with 100 μgwild-type LST, F11 or PBS, administered as a single bolus intravenousinjection. The survival rate for both protein treatments was 1/3. The100 μg dosage was selected because wild-type LST is known to be onlypartially efficacious at this dose. By using this dose, clear efficacyequivalence for the F11 variant could be demonstrated. This analysisindicated that both proteins had equivalent efficacy in vivo (FIG. 8).

The immunogenicity of the F13 variant was compared to wild-type LST inDR4 HLA transgenic mice, i.e., mice bearing partially humanized immunesystems. 100 μg of wild-type LST or variant F13 were mixed with completeFreund's adjuvant and injected subcutaneously into DR4 mice. Fourteendays later, antibody titers were measured by ELISA. Variant F13 yieldedmore than 200-fold lower antibody titers compared to wild-type LST. Toassess the impact of antibody titers on antibacterial efficacy, the micewere challenged with an intraperitoneal injection of 2×10⁸ MRSA strainUSA400, and 1 hour later the mice were treated with 500 μg wild-typeLST, variant F13, or PBS, administered as a single bolus intravenousinjection. Both mice receiving PBS sham treatments died as did both micetreated with wild-type LST. In contrast, both mice treated with variantF13 survived. Thus, the reduced immunogenicity of F13 conferred enhancedtherapeutic efficacy in vivo.

In a similar set of experiments, the immunogenicity and efficacy ofvariant F12 was compared to wild-type LST in the absence of adjuvantusing DR4 HLA transgenic mice. At week 0, mice were challenged with anintraperitoneal injection of 2×10⁸ MRSA strain USA400, and 1 hour laterthe mice were treated with 500 μg wild-type LST or 500 μg variant F12,given as a single bolus subcutaneous injection. Subsequently, mice werechallenged weekly with an intraperitoneal injection of 1×10⁹ MRSA strainUSA400, and treated as above. Antibody titers were measured weeklybeginning at week 2. Wild-type LST was able to rescue mice from a totalof four recurrent, systemic, MRSA infections, but failed to rescue anymice from the fifth infection (Table 23). Variant F12 rescued all micefrom four recurrent, systemic, MRSA infections (Table 24). Thesubstantially lower antibody titers elicited by variant F12 indicatethat this variant is more efficacious than wild-type LST.

TABLE 23 # Mice # Treated # Mice # Sham # Mice Treated Mice TreatedTreatment Week Infected with Enzyme Surviving with PBS Surviving 0 7 7 70 NA 1 7 6 6 1 0 2 6 5 5 1 0 3  5* 4 4 1 0 4 4 3 0 1 0 *ELISA antibodytiters measured for only four representative mice out of five total.

TABLE 24 # Mice # Treated # Mice # Sham # Mice Treated Mice TreatedTreatment Week Infected with Enzyme Surviving with PBS Surviving 0 10 1010 0 NA 1 10 9 9 1 0 2  9* 8 8 1 0 3  8 7 7 1 0 4  7 6 6 1 0 5 pendingpending pending pending pending *ELISA antibody titers measured for onlyeight of nine total.

What is claimed is:
 1. A deimmunized lysostaphin comprising a mutationat one or more of Ser124, Ser122, Asn121, Arg118, Ile99, Lys95, Tyr93,Leu83, Lys46, Ile41, Asn13, or Asn12 of SEQ ID NO:49.
 2. The deimmunizedlysostaphin of claim 1, wherein said lysostaphin is aglycosylated. 3.The deimmunized lysostaphin of claim 1, wherein the mutation comprisesSer124Gly, Ser122Asp, Asn121Gly, Arg118Thr, Ile99Gln, Lys95Glu,Tyr93His, Leu83Met, Lys46His, Ile41Glu, Asn13His, Asn12Gly, or acombination thereof.
 4. The deimmunized lysostaphin of claim 1, furthercomprising one or more amino acid substitutions in the C-terminalbinding domain.
 5. A pharmaceutical composition comprising thedeimmunized lysostaphin of claim 1 and a pharmaceutically acceptablecarrier.
 6. The pharmaceutical composition of claim 5, furthercomprising an antibiotic.
 7. The pharmaceutical composition of claim 6,wherein said antibiotic comprises a β-lactam, cephalosporin,aminoglycoside, sulfonamide, antifolate, macrolide, quinolone,glycopeptide, polypeptide or a combination thereof.
 8. A method forpreventing or treating a microbial infection comprising administering toa subject in need of treatment the pharmaceutical composition of claim5, thereby preventing or treating the subject's microbial infection. 9.The method of claim 8, wherein said infection is a bacterial infection.10. The method of claim 9, wherein said bacterial infection is caused bybacteria from the genus Staphylococcus.